Hsp90绑定磷脂特性及其对肌内磷脂水解的影响机制

Lipolysis of phospholipids produces a large number of unsaturated fatty acids, a process that is highly correlated with lipid oxidation and generation of volatile flavor compounds in meat products. Therefore it is essential to study the impact factors involved in the process of intramuscular phospholipid lipolysis. Applicant's previous evidence suggested that heat shock protein 90 (Hsp90) could form a stable complex with neutral phospholipid and markedly reduce phospholipase A2 mediated phospholipid lipolysis, we consequently infer that the phospholipid binding Hsp90 could restrain phospholipid lipolysis. To clarify the protection role of Hsp90 upon phospholipids, duck meat will be served as a vehicle, the X-ray diffraction and Atomic Force Microscope methods will be applied to investigate the microstructure profile of Hsp90-phospholipid complex and their impact factors; To reveal the correlation between Hsp90 and phospholipid lipolysis, the changes in the phospholipid lipolysis products will be tracked under dual environments - the aqueous simulation system and the intramuscular simulation system, assisted with high-performance liquid chromatography and tandem mass spectrometric (HPLC-MS/MS) and gas chromatography (GC). This project is devoted to provide a new approach for studies on meat flavor, as well as a theoretical basis for in depth perception of meat quality control during preservation and processing.

肌内磷脂水解会产生大量不饱和脂肪酸，与肉品脂质氧化和挥发性风味物质形成关系密切，因此肌内磷脂水解过程的影响因素研究十分重要。申请者前期研究证实，Hsp90可与中性磷脂结合成稳定的绑定体，可以显著削弱磷脂酶A2对磷脂的水解作用，据此推测"Hsp90的磷脂绑定特性能够抑制肌内磷脂水解"。本项目以鸭肉为研究载体，采用X射线衍射分析仪和原子力显微镜（AFM），探究鸭肉中Hsp90绑定磷脂的微观结构特征和影响因素，推测Hsp90的磷脂保护效应；再运用液质联用（HPLC-MS/MS）、气相色谱（GC）等手段，分析水相和鸭肉内环境模拟反应体系中磷脂水解产物的变化规律，双重角度验证Hsp90对肌内磷脂水解的影响，明确Hsp90与磷脂水解的相关性。项目成果不仅为肉制品风味形成机理研究提供了新思路，也为深度挖掘肉品贮藏和加工过程中的品质调控方法提供理论依据。

以鸭肉为载体，对（热休克蛋白90）Hsp90进行分离纯化；采用荧光光谱、红外光谱和表面等离子共振分析，探究鸭肉中Hsp90绑定磷脂的微观结构特征和影响因素，推测Hsp90的磷脂保护效应；再运用高效液相色谱-蒸发光散射、气相色谱等手段，分析水相和肌肉内环境模拟反应体系中磷脂水解产物的变化规律，明确Hsp90与肌内磷脂水解、肉品品质的相关性。结果表明，鸭肉中Hsp90表观分子量为84.4 kD；磷脂与Hsp90的结合作用力主要是氢键和范德华力，磷脂的极性头部和脂肪酸链都可与Hsp90产生稳定的结合，且Hsp90对磷脂的结构产生影响；ATP、pH和Cl-等因素对Hsp90的磷脂绑定量影响较大；Hsp90的C、M（中间）、N三段在结合磷脂时显示不同的亲和力，C-端片段强结合、N-端片段中等强度结合、M-片段弱结合，其中Hsp90-C端片段能显著抑制磷脂的水解和氧化；Hsp90含量与肉品的pH值、保水性和游离脂肪酸含量显著相关，高pH组的Hsp90含量最高、游离脂肪酸含量最低且保水性最强，而低pH组的Hsp90含量最低、游离脂肪酸含量最高且保水性最弱，一定程度上反映了Hsp90与细胞膜磷脂结合能减少游离脂肪酸的产生，从而保护细胞膜的完整性有利于保水性提高。项目成果不仅为肉品风味形成机理研究提供新思路，也为深度挖掘肉品脂质氧化控制方法奠定理论基础。