为探究凡纳滨对虾磷脂（PL）在贮藏过程中的水解机理，本研究建立了磷脂相关酶与磷脂酰胆碱（PC）的体外模型，模拟凡纳滨对虾磷脂水解反应。实验分别从凡纳滨对虾体内分离纯化得到磷脂相关酶与PC，进行体外反应，并采用“鸟枪”脂质组学法及气相色谱—质谱法分析水解反应前后PC、溶血磷脂（LPL）及游离脂肪酸（FFA）的组成和含量变化，进而推断磷脂的水解机理。结果显示，凡纳滨对虾体内磷脂水解酶包括脂肪酶、磷脂酶A₁（PLA₁）、磷脂酶A₂（PLA₂）、磷脂酶C（PLC）及磷脂酶D（PLD），其中PLA₂活力最高，可达177.87 U。体外模拟反应中，凡纳滨对虾PC含量由516.45显著降至146.14 mg/g，总FFA含量由36.42上升至568.57 mg/g，其中多不饱和脂肪酸（PUFA）显著增加了280.5 mg/g。通过相关性分析水解前后磷脂水解酶活力变化与PC含量变化，发现PC的变化与PLA₂显著相关（R=0.91）。研究表明水产品中PC变化是磷脂相关酶催化的水解反应，其水解产物主要为FFA与LPC，且PLA₂对水产品贮藏过程中磷脂的水解影响最大。本实验建立了新型的体外水解模型，并初步探究了磷脂的水解机理，为水产品贮藏及磷脂酶的研究及应用奠定了理论基础。

Litopenaeus vannamei contains abundant phospholipids (PL), especially rich in polyunsaturated fatty acids (PUFA), which was easy to be hydrolyzed during storage and to produce free fatty acids (FFA). The increase of FFA leads to flavor, color and other changes, which affects the quality of aquatic products. Therefore in recent years, some information regarding the changes of lipids in aquatic food during handling and storage has been reported, such as L. vannamei, Pacific white shrimp and so on. However, the limited information on PL hydrolysis mechanism during storage is available, which is mainly owing to the complex system of PL hydrolysis and the lower content of phospholipases and lipase in aquatic food than mammals and microbes. Fortunately, electrospray ionization tandem mass spectrometry (ESI-MS/MS) has demonstrated high accuracy and reproducibility in phospholipids analysis, which brings potentiality for the study of the PL hydrolysis. Thus, to better understand the hydrolysis mechanism in sea foods during storage, we established a model system of phospholipids hydrolysis for the first time, which avoided the barrier of the complex system. We had extraction and purification of the phospholipases and PC from L. vannamei, and measured the enzyme activity of purified phospholipases. What's more, a fast and efficient "shotgun" lipidomics strategy was applied to analyze the levels and changes of PL, lysophosphatides (LPL) in L. vannamei. And the content of free fatty acids was analyzed by gas chromatography-mass spectrometry (GC-MS). The results showed that the phospholipases extracted from L. vannamei included lipase, phospholipase A₁ (PLA₁), phospholipase A₂ (PLA₂), phospholipase C (PLC) and phospholipase D (PLD), among them PLA₂ had the highest enzyme activity. And in the reaction system, the content of PC decreased from 516.45 to 146.14 mg/g. Meanwhile, FFA had a significant increase from 36.42 to 568.57 mg/g, and PUFA increased by 280.5 mg/g. Furthermore, the study showed the close correlation between PC hydrolysis and PLA₂ (R=0.91) by measuring the enzymes activities before and after hydrolysis reaction. Besides, PLA₁ also showed the relatively close relationship with the PC hydrolysis compared to PLA₂. These information, mentioned above, suggested that aquatic phospholipases can hydrolyze phospholipids and produce a series of hydrolyzates, mainly including LPL and FFA. In a word, this study indicated the hydrolysis mechanism of phospholipids in aquatic products was correlated with the activities of lipid hydrolytic enzymes during storage, and PLA₂ was crucial to the hydrolysis of PC, which laid a theoretical foundation for the aquatic food storage and phospholipids research. Therefore, the suppression of lipid hydrolysis and the study of the complete hydrolysis mechanism will be a means to maintain the quality of aquatic food stored in ice, which may be a significant research direction in the future.

国家自然科学基金（31330060）；“泰山学者”攀登计划