基于光谱技术的维生素B12与大豆分离蛋白相互作用分析

Spectroscopy Analysis of Interaction between Vitamin B12 and Soybean Protein Isolate

为了提高维生素B12稳定性和开发维生素B12营养强化食品,以维生素B12-大豆分离蛋白复合体系为研究对象,利用光谱技术(荧光光谱、紫外-可见吸收光谱、红外光谱、圆二色谱)分析维生素B12与大豆分离蛋白的相互作用对蛋白质结构的影响。荧光光谱分析表明,随着维生素B12质量浓度的增大,大豆分离蛋白的荧光强度不断降低,通过Stern-Volmer方程计算可得,维生素B12对大豆分离蛋白的猝灭方式属于静态猝灭,二者主要通过范德华力和氢键作用结合,结合位点数为1;同步荧光光谱分析表明,维生素B12与大豆分离蛋白结合位点位于色氨酸附近;紫外光谱分析表明,维生素B12诱导色氨酸残基附近的微环境疏水性增强,蛋白分子的结构发生改变;红外光谱和圆二色谱分析表明,维生素B12的加入导致大豆分离蛋白的二级结构发生改变,具体表现为α-螺旋和β-转角相对含量增加、β-折叠和无规则卷曲相对含量减少。

The vitamin B12 is an essential vitamin and participates in the biochemical reaction of the human body,including folic acid conversion and methionine synthesis.However,it is easy to lose its nutrition and function in the food processing process and storage such as light,heating and pH value.In order to improve the stability of the vitamin B12 and the vitamin B12 nutrition fortified food,the interaction between vitamin B12 and soybean protein isolate and the effect of vitamin B12 on protein structure were studied by using spectroscopic techniques(fluorescence spectrum,ultraviolet spectrum,infrared spectrum and circular dichroism spectrum).The result of fluorescence spectrum showed that the fluorescence intensity of soybean protein isolate was decreased with the increase of vitamin B12 concentration.The quenching mode of vitamin B12 on soybean protein isolate was static quenching by calculating the Stern-Volmer equation.Soybean protein isolate and vitamin B12 were bound by Van der Waals force and hydrogen bonding and binding site was one.Synchronous fluorescence spectrum indicated that the binding site of vitamin B12 and soybean protein isolate was located near tryptophan.Ultraviolet absorption spectrum presented that vitamin B12 enhanced hydrophobicity of the microenvironment near the tryptophan residue and changed the structure of protein molecules.The results of infrared spectrum and circular dichroism spectrum showed that the secondary structure of soybean protein isolate was changed with the addition of vitamin B12,which represented that the contents ofα-helix andβ-rotation were increased,and the contents ofβ-fold and irregular curl were decreased.