酶法脱酰胺协同构象保护改善肌原纤维蛋白水溶液稳定性的机制研究

One major functional challenge hindering the development of muscle protein-based beverages is that myofibrillar proteins (MP) usually show poor physical stability in aqueous solution. Enzymatic deamidation can increase the protein colloidal stability, when synergized with protecting protein conformation by sucrose, it may have great potential of enhancing protein stability in water. This project initially intends to verify the feasibility of this strategy by studying the response of storage and thermal stability to different enzymatic modification and sucrose addition conditions. Based on the above results, the molecular composition, structure and morphology, and colloidal properties of modified proteins will be characterized by using circular dichroism, atomic force microscopy, dynamic/static light scattering, etc. In addition, attenuated total reflection fourier transform infrared, gel electrophoresis-peptide mapping mass spectrometry, synthetic peptide verification, etc., will be adopted to determine the modification sites of myosin tail, aiming at revealing the dynamic process of myosin thick filament assembly as well as clarifying the reasons for enhanced storage stability of MP aqueous solution. Differential scanning calorimetry, steady shear/dynamic oscillating rheology, peptide mass spectrometry, etc., will be applied to study protein conformational preservation domains, the change of aggregates, molecular forces and thermal aggregation behavior, thereby trying to elucidate the mechanism on improving the thermal stability of MP aqueous solution. The results from this project will provide technical support and theoretical basis for the reasearch and development of new muscle protein-based beverages.

肌原纤维蛋白（MP）水溶液的物理稳定性差是制约肌肉蛋白在流体食品中应用的关键屏障。酶法脱酰胺作用可提高蛋白胶体稳定性，协同蔗糖对蛋白构象的保护，具有改善其水溶液稳定性的可行性。本项目拟通过研究MP水溶液贮藏和热稳定性对不同酶法修饰和蔗糖添加条件的响应规律，以验证该策略的可行性；在此基础之上，运用圆二色谱、原子力显微镜、动/静态光散射等技术对修饰蛋白的分子组成、结构与形貌、胶体特性进行表征，同时借助全反射傅里叶红外光谱、凝胶电泳–肽谱图全序列质谱鉴定、合成肽段验证分析等技术探究肌球蛋白尾部修饰位点，揭示肌球蛋白粗丝装配动态过程，阐明MP水溶液贮藏稳定性的增强机制；应用差式扫描量热、稳态剪切/动态震荡流变、肽段质谱鉴定等技术研究蛋白特征构象保护域、聚集体片段、作用力变化与热凝聚行为，以解析MP水溶液热稳定性增强机制。本项目的开展可为开发新型肌肉蛋白产品提供技术支持和理论基础。

肌肉蛋白因其营养和结构特性，被公认为是开发新型蛋白产品的良好原料。在骨骼肌肌肉蛋白中，肌原纤维蛋白（Myofibrillar protein，MP）是其主要组分，MP在低盐条件下的加工特性缺陷限制了其新型产品的开发与应用（如流体食品、软化食品等）。这其中，如何实现MP水溶解、提高热稳定性和拓展功能等问题在肉类科学领域备受关注，开展相关的基础研究成为亟需完成的任务。本项目以此为出发点，创新式引入食品酶法脱酰胺技术，系统研究了谷氨酰胺酶（Protein-glutaminase，PG）脱酰胺作用对MP在水溶液中的胶体稳定性的影响，通过探究蛋白多尺度结构和肌球蛋白粗丝装配对PG脱酰胺作用的响应规律，揭示了PG实现MP水溶的分子机制。随后，探讨了MP胶体热特性变化规律，在此基础上，建立了新型加工策略以抑制MP热聚集，提高其热稳定性，阐明了MP胶体热稳可调的分子机制。最后，借助PG精细化脱酰胺，探究了实现MP可逆凝胶化的最佳处理条件，明确了关键因素。其重要结果分述如下：（1）PG可定向调控MP内源电荷，诱发肌纤维解离，脱酰胺16h后，脱酰胺度为6.54%，MP水溶性由19%提升到64%，脱酰胺处理不会引发氨基酸的营养价值损失和蛋白亚基水解；（2）PG脱酰胺作用可以减弱尾部区域带电团簇的正电性，推测可影响肌球蛋白在低离子强度条件下的二聚体规则组装，从而抑制其粗丝的动态装配，进而提升MP的水溶性；（3）没食子酸介导的MP可溶性低聚物可实现胶体热稳可调。没食子酸碱性氧化作用可诱发MP发生结构重排，形成可溶解复合粒子，分子量约为1225kDa，推测主要为肌球蛋白二聚体。该复合粒子具有优异的物理稳定和粘度可调特性，因与胃蛋白酶pepsin的亲和力下降，表现出消化迟滞行为；（4）PG的精细脱酰胺可诱导MP产生可逆凝胶化特性，此类凝胶与蛋白单α-螺旋和双螺旋卷曲之间的可逆组装密切相关。综上所述，PG脱酰胺是拓展和调控MP低盐功能特性的有效策略，低盐水溶与可逆凝胶化的实现能够为新型肌肉蛋白食品的开发提供思路和理论依据。