外源性羟基自由基氧化对银杏果分离蛋白凝胶特性的影响

Effect of exogenous hydroxyl radical-induced oxidation on gelation properties of ginkgo seed protein isolate

摘要:

采用Fenton反应（0.016 g/L FeCl3 + 0.018 g/L抗坏血酸 + 0-0.680 g/L H2O2）模拟羟基自由基生成体系，通过对银杏果分离蛋白（GSPI）进行氧化，以制备GSPI溶胶和热诱导凝胶，基于物化分析法、聚丙烯酰胺凝胶电泳、SEM、拉曼光谱等测试和表征，探究含不同浓度羟基自由基氧化对GSPI物化特性及其热诱导凝胶的质地、水合性质、微观结构和二级结构的影响。结果表明，羟基自由基存在下，GSPI表面的疏水性氨基酸残基暴露，巯基氧化形成分子内或分子间二硫键，疏水相互作用和二硫键的变化会导致GSPI不同程度的展开和交联，适当的氧化（H2O2质量浓度0.034~0.340 g/L）能够引起α-螺旋去卷积，β-折叠增加，促进蛋白质分子交联，形成纠缠凝胶网络，提高蛋白凝胶的机械强度、流变学性能。当H2O2质量浓度0.340 g/L时，GSPI凝胶的硬度、胶黏性和咀嚼性分别达到最大，与未氧化（H2O2质量浓度0 g/L）的凝胶相比分别增大至2.08、2.53和3.01倍。而过度氧化（H2O2质量浓度≥0.680 g/L）虽然会提高GSPI凝胶持水性能，但会导致蛋白分子中氢键被破坏，产生过量的二硫键，阻碍其它活性基团的相互作用，抑制凝胶网络的形成，降低凝胶强度。

Abstract:

Fenton reaction, which contains 0.016 g/L FeCl3 + 0.018 g/L ascorbic acid + 0-0.680 g/L H2O2, was used to as a hydroxyl radical generation system, and then to oxidize ginkgo seed protein isolate (GSPI) to prepare their protein sols and heat-induced gels. The effect of hydroxyl radical concentration on the physiochemical properties of GSPI and subsequently its heat-induced gelation properties were investigated. Physicochemical analysis, SDS-PAGE, SEM, and Raman spectroscopy were used to characterize the protein sols and gels for their physicochemical properties, hydration properties, microstructure, and secondary structure. The results indicated that oxidation exposed hydrophobic amino acid residues on the surface of proteins and promoted sulfhydryl groups to form intramolecular/intermolecular disulfide bonds. Hydrophobic interactions and disulfide bonds resulted in varying degrees of protein unfolding and cross-linking. Moderate oxidation (e.g.: 0.034~0.340 g/L H2O2) caused α-helix deconvolution. The increase of β-sheets promoted the protein crosslinking, formed an entangled gel network, and consequently improved the mechanical strength and rheological properties of resulting protein gel. As H2O2 concentration increased to 0.340 g/L, the hardness, gumminess, and chewiness of the GSPI gel increased to 2.08, 2.53, and 3.01 folds, respectively, compared with the unoxidized gel (0 g/L). Excessive oxidation (e.g.: H2O2 concentration > 0.680 g/L) improved water holding capacity of the gels, whereas the oxidation impaired hydrogen bond formation, formed excessive disulfide bonds, hindered the interaction of other active groups, inhibited the formation of gel networks, and reduced gel strength.