漆酶催化大米蛋白与阿魏酸交联及其结构和抗氧化活性的变化

Laccase-mediated Cross-linking of Rice Protein with Ferulic Acid: Changes in Structural Properties and Antioxidant Activity

摘要:

本文研究了大米蛋白（Rice protein，RP）与阿魏酸(Ferulic acid，FA)的酶法交联及其在水相和乳液中的抗氧化能力，通过HPLC分析FA的含量以及SDS-PAGE、紫外光谱、红外光谱和扫描电子显微镜分析RP的结构变化。HPLC分析表明FA易被漆酶催化氧化，且FA的氧化产物与RP的亚基反应，从而引起大米蛋白分子量的增加；随着FA浓度的增加，RP的紫外图谱在300~350 nm处的吸收逐渐增加，交联样品呈现橘黄色，说明氧化阿魏酸能共价结合大米蛋白；RP与FA经酶法交联后RP二级结构发生变化，α-螺旋和β-转角含量分别降低了13%与2%，无规卷曲和β-折叠含量分别提高了11%与4%，蛋白的显微结构从聚集变为松散；FA与RP交联产物的清除DPPH自由基能力和清除ABTS自由基能力分别从32.31%增加到71.43%和从3.34%增加到77.61%，FA与RP交联产物可显著降低乳液中氢过氧化值、TBARS值、己醛的含量，说明交联可有效提高大米蛋白的抗氧化活性。

Abstract:

The enzymatic cross-linking of rice protein (RP) and ferulic acid (FA) and the antioxidant activity of the resultant product in the aqueous phase and emulsion were studied, and the FA content was investigated by high performance liquid chromatography (HPLC). Changes in the structural properties of RP were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet (UV) spectroscopy, Fourier transformed infrared (FT-IR) spectroscopy, and scanning electron microscopy. HPLC results showed that the FA easily underwent laccase-catalyzed oxidation, and the oxidation product could react with the subunits of RP, resulting in an increase of RP protein molecular weight. With increasing FA concentration, the absorbance band between 300 and 350 nm in the UV/visible spectra of RP gradually increased, and the cross-linked products had an orange-yellow color, indicating that the oxidation products bound covalently to the free amino groups of RP. After the enzymatic cross-linking of RP and FA, the secondary structure of RP changed, the contents of β-turn and α-helix were reduced by 13% and 2%, respectively, the contents of random coil and β-sheet were increased by 11% and 4%, respectively, and the microstructure of RP became loosened from the aggregated state. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging abilities of the RP-FA cross-linked product were increased from 32.31% to 71.43% and from 3.34 % to 77.61%, respectively, compared to those of RP. RP-FA cross-linked product could significantly reduce the level of hydroperoxide, 2-thiobarbituric acid reactive substances (TBARS), and hexanal in the emulsion, suggesting that cross-linking of RP can effectively enhance the antioxidant activity of RP.