制备方法对乳清分离蛋白-绿原酸共价接枝物结构和功能性质的影响

Effect of Preparation Methods on the Structural and Functional Properties of Whey Protein Isolate-Chlorogenic Acid Conjugates

目的:研究制备方法对乳清分离蛋白(whey protein isolate,WPI)-绿原酸(chlorogenic acid,CA)共价接枝物结构和功能性质的影响。方法:分别以碱法、自由基法和酶法制备WPI-CA共价接枝物,对其反应基团含量、CA接枝量、分子结构、表面疏水性、热稳定性、抗氧化活性和乳化性等进行分析。结果:游离氨基、色氨酸和巯基均参与了反应,游离氨基为主要反应基团。碱法、自由基法和酶法接枝物的CA接枝量分别为(52.70±1.81)、(42.57±1.85)μmol/g和(63.75±2.50)μmol/g。CA的共价接枝使WPI二级结构中α-螺旋相对含量减少,无规卷曲结构相对含量增加,分子内源荧光强度降低。碱法和酶法接枝会降低WPI表面疏水性,增强其热稳定性,自由基法的影响则与两者相反。与WPI相比,WPI-CA共价接枝物的抗氧化活性和乳化性显著增强(P<0.05),且抗氧化活性与CA接枝量成正比。结论:酶法接枝效率最高,且所得WPI-CA接枝物热稳定性和抗氧化活性最强。本研究可为蛋白质-多酚共价接枝物类抗氧化性载体材料的制备及应用提供参考。

Objective:To evaluate the effect of preparation methods on the structural and functional properties of whey protein isolate(WPI)-chlorogenic acid(CA)conjugates.Methods:The methods tested were an alkali method,a free radical method and an enzymatic method.The reactive group content,CA amount of covalent binding,molecular structure,surface hydrophobicity,thermal stability,antioxidant activity and emulsion properties of WPI-CA conjugates were analyzed.Results:Free amino groups,tryptophan and sulfhydryl groups all participated in the grafting reaction with free amino group being the major reactive group.The CA amounts of covalent binding of the conjugates prepared by the alkali,free radical and enzymatic methods were(52.70±1.81),(42.57±1.85),and(63.75±2.50)μmol/g,respectively.The conjugates showed lessα-helix relative content,higher random coil relative content and lower fluorescence intensity than WPI.Covalent bonding of CA induced by the alkali and enzymatic methods decreased the surface hydrophobicity and increased the thermal stability of WPI,while the free radical method led to the opposite results.The emulsion properties and antioxidant activity of the conjugates were higher than that of WPI,and the antioxidant activity was positively proportional to the amount of bonded CA.Conclusion:The highest grafting efficiency was obtained by the enzymatic method,and the prepared conjugates showed best thermal stability and antioxidant activity.This study can provide a reference for the preparation and application of protein-polyphenol conjugates as antioxidant carrier materials.