骨骼肌肌球蛋白高压变性和成胶机理研究

High pressure processing can assist or replace of traditional heating manipulation, and produce the gel-type meat products with many excellent attribute such as safety, reduced loss of nutritions, and without too much addition of the salt sodium and fats. However, the lack of information about high-pressure induced denaturation and gelation mechanism of myosin restricted the use of it. In this project, myosin from rabbit skeletal muscle is used as material. The effects of high pressure operation for pre-rigor muscle, ionic strength, pH, content of NaCl, phosphates and fat on the myosin gelation properties in two processing conditions (high pressure assisted gelation or high pressure gelation) are investigated by tested the properties of TPA, dynamic rheology and water holding capacity of myosin gels. Then, the changes of the biochemical properties of myosin and its main subfragments, including functional groups exposed on the surface, ATPase activities and the tertiary and quaternary structures are determined as well as the thermal factors transformation and the dynamic process of aggregating during heating. The main equipments or techniques are enzyme-labeled instrument, Raman spectroscopic analysis, the dynamic and captive laser scattering, particle size analyzer and differential scanning calorimetry (DSC). The chemical bonds types during gelation are determined using specific chemical modifiers. The main structure sites which binding each other to from the gel, and the important subfragments of myosin molecule contribute to the gel forming and the water holding capacity are studied by tested the subjects of enzyme degradation, SDS-PAGE, HPLC and LS-MS. Finally, the dynamic models of the high pressure assisted or high pressure induced myosin gelation are established based on the theory of "Fractal". The theory of high pressure modification of myosin, and the mechanisms of high pressure induced myosin denaturation and gelation will be elucidated through the contributions of molecular structure and conformational aspects, chemical forces bonds and kinetics of gelation. So the research results of this task will provide theoretic guidelines and technical supports for the R&D of high pressure gel-type meat products.

高压处理可以辅助或替代热加工，生产安全性高、营养损失小、食盐和脂肪添加量少的凝胶类低温肉制品。但是肌球蛋白高压变性和成胶机理还未阐明，极大限制了该新技术的应用。本课题以兔肉骨骼肌肌球蛋白分子为实验对象，研究僵直前高压处理、离子强度、pH值、食盐、磷酸盐和脂肪添加量对高压辅助凝胶和高压凝胶两种工艺条件下，肌球蛋白凝胶特性的影响；在此基础上，利用酶标仪、拉曼光谱仪等测定表面官能团含量以及分子构象变化；利用激光光散射仪、粒度仪和差示热量扫描仪测定高压聚集和热力学变化；利用化学修饰法测定化学作用力对凝胶形成的贡献；利用酶解、SDS-PAGE、HPLC和质谱等技术测定参与凝胶形成的关键结构域、对凝胶结构和保水性起重要作用的分子片段；最后利用分形研究凝胶形成动力学过程。以期揭示高压修饰的作用规律，阐明肌球蛋白高压变性和凝胶形成机理，为高压凝胶类肉制品的开发提供理论指导和技术支持。

超高压加工作为非热加工新技术能够显著改善凝胶类肉制品的品质，但是超高压对肉中蛋白质的修饰及其对凝胶功能特性的影响还未彻底阐明，限制了其进一步应用。本课题通过建立两个模型体系对此进行探究，以期为超高压的应用提供理论参考。.模型系统-1（模-1）以僵直前兔腰大肌为原料，对其进行100-300MPa保压15s或180s处理，未高压组为对照。随后进行加盐斩拌成兔肉糜，研究超高压对模-1中蛋白质的变性修饰及凝胶功能特性的影响。模型系统-2（模-2）以加盐斩拌的兔肉糜为原料，对其进行100-300MPa保压3、9或15min的处理。研究超高压对模-2中蛋白的变性修饰及凝胶功能特性的影响。.研究发现，200MPa15s的超高压处理能够显著提高模-1的蛋白质凝胶功能特性，如保水性、质构特性、以及流变性质等。模-2在200MPa 9min的超高压处理后，能显著改善肌球蛋白凝胶功能特性。理化指标测定结果表明超高压处理诱导蛋白解折叠，同时伴有α-螺旋降低、β-折叠增加的二级结构变化现象。通过对凝胶微观结构的分型维值以及孔隙度的分析中发现，上述超高压条件处理后形成的热凝胶分型维数以及空隙度均高于对照组，说明形成的凝胶网络更为复杂。.为进一步探究机理，在模-1和模-2的基础上提取肌球蛋白进行研究。结果证明，超高压通过改变肌球蛋白的理化特性，如蛋白的空间构像、二级结构以及蛋白分子之间的相互作用达到改变凝胶特性的作用。研究中还发现超高压具有诱导肌球蛋白形成二聚体的特异性，同时蛋白空间结构发生膨胀，改变蛋白-蛋白之间的交互作用。这些超高压诱导的变化通过改变凝胶微观结构，进一步影响凝胶的宏观功能特性。利用分子动力学模拟探究超高压过程中肌球蛋白分子的变化，结果证明超高压通过将水分子“迫进”蛋白空间结构中的方式促使蛋白的空间构像解折叠，同时改变蛋白间的相互作用和二级结构，表现为疏水基团和氢键作用比例增加以及α-螺旋含量的减少。这些变化是肌球蛋白形成二聚体的关键。.上述结果阐明了超高压诱导肌球蛋白变化对凝胶功能特性影响的关键因素，为超高压技术在凝胶类肉制品生产加工领域的应用奠定了坚实的理论基础。