强碱诱导的皮蛋蛋白三维链式共聚凝胶“碱伤液化”机制及其阻控研究

It is still necessary to add heavy metal compounds such as CuSO4 in order to prevent the “alkali injury liquefaction” of the preserved egg white gel. However, the mechanism of “alkali injury liquefaction” of preserved egg white gel is not clear, which hinders the improvement of its non-heavy metal technology. Therefore, this research intends to build a pickled model of “alkali injury liquefaction” in the eggshell, the transformation behavior of preserved egg gel during “alkali injury liquefaction” will be studied using rheometer, texture analyzer, SEM, SAXS as well as other physical and microstructure analysis methods. At the same time, modern instruments and chemical analysis will be used to study the evolution of protein structure and molecular force, and reveal the law of its liquefaction behavior. Further, strong alkali-induced egg white “sol-gel-sol” transformation model outside the shell will be built to study the single protein and its combination in gel assembly. The changes of protein molecular conformation, protein degradation and disintegration of force during the process of liquefaction will be revealed and the protein cracking sites will be clearly defined. The relationship of alkali response will be explored and a dynamics model of liquefaction will be established, which illuminate the mechanism of “alkali injury liquefaction” of three-dimensional chained copolymerized gel in preserved egg white. Resistance control method of “alkali injury liquefaction” of preserved egg white gel will be screened and optimized based on synergism of basic pickled condition and additives. And then their resistance control technology system will be built, which will provide theoretical basis for quality control and technology improvement of preserved egg.

为避免强碱诱导的皮蛋蛋白凝胶“碱伤液化”，其腌制液中仍必须添加硫酸铜等重金属化合物。然而尚不明确的“碱伤液化”机制阻碍了其无重金属化工艺的改进。因此，本项目拟构建壳内“碱伤液化”腌制模型，借助流变仪、质构仪、SEM和SAXS等物性与微观结构分析手段研究皮蛋蛋白凝胶“碱伤液化”的物态转变行为；同时运用现代仪器和化学分析手段研究蛋白质结构与分子作用力的演化规律。进一步构建壳外强碱诱导蛋清蛋白“溶胶-凝胶-溶胶”转变模型，对蛋清和参与皮蛋凝胶组装的单一蛋白质及其组合进行研究，揭示液化过程中蛋白质分子构象转变、蛋白质降解、作用力瓦解等规律，明确蛋白质裂解位点，并探讨碱响应关系，建立液化动力学模型，从而解析皮蛋蛋白三维链式共聚凝胶的“碱伤液化”机制。在此基础上，筛选并优化腌制基础条件协同添加物抑制皮蛋蛋白凝胶“碱伤液化”的方法，构建其阻控技术体系，为皮蛋加工过程中品质控制与工艺改进提供理论基础。

为避免皮蛋蛋白凝胶“碱伤液化”，其腌制液中仍须添加硫酸铜等重金属化合物，这已成为制约其产业进一步发展的瓶颈。针对皮蛋蛋白凝胶“碱伤液化”机制不明确的现状，本项目通过运用多种现代仪器和化学分析方法研究壳内外蛋清蛋白凝胶在液化过程中凝胶特性、流变特性、微观结构等物化行为，并深入系统研究了蛋清蛋白质构象变化、蛋白质降解以及分子作用力的演化；并在此基础上，筛选并优化腌制条件协同添加物抑制皮蛋“碱伤液化”的有效方法。结果表明，强碱诱导的蛋清蛋白凝胶在经历“碱伤液化”过程时的硬度、表面疏水性和释放肽段数均呈下降趋势，蛋白凝胶逐步转变为具有剪切稀化特性的溶胶状态，这一过程与碱液浓度呈现显著的正相关关系。红外光谱显示液化过程中蛋白质二级结构的β-折叠随α-螺旋的增加而降低，而β-折叠的减少是不利于凝胶形态稳定的。此外，液化过程中二硫键不断断裂，非共价交联逐渐减弱，体系中离子键含量迅速升高，变性的蛋白质分子暴露出更多的游离基团，蛋白质之间的相互作用进一步瓦解。在不同碱度诱导不同时间的碱伤液化体系下，使用LC-MS/MS分析得到了不同的肽段数和更多特异性的肽段序列，证实了“碱伤液化”过程中蛋白质持续降解为多肽和氨基酸，且主要表现为无序性降解，在整个液化过程中仅发现了SVLMAMGITDVF 和AVHAAHAEINEAGREVV两个潜在的共同肽段。在液化机制阐明的基础上，通过筛选明确抗坏血酸钠添加量大于2%时，可以明显抑制无重金属皮蛋蛋白凝胶的“碱伤液化”作用。在相同腌制时间内，皮蛋蛋白的pH值会随着抗坏血酸钠浓度的增加而下降，并且皮蛋蛋白棕色会随着抗坏血酸钠浓度的增加而加深，说明抗坏血酸钠对皮蛋蛋白凝胶的维持与色泽均具有有利影响。项目成果可为皮蛋加工过程中品质控制与无重金属化工艺改进提供理论基础。在本项目支持下共发表了SCI收录论文14篇，其中中科院一区论文12篇、二区论文1篇、三区论文1篇。

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