Melanization is the major deterioration in organoleptic quality of shrimp Litopenaeus vannamei during cold storage and leads to dramatical reduction of commercial value. So far, the biochemical cascade mechanism triggering and accelerating melanosis progression remains unclear. Herein, this investigation aimed to monitor the melanosis development in L. vannamei during 5 days’ cold storage at 4 °C and to explore the role of serine protease (SP) in zymogen activation of polyphenol oxidase (PPO) by application of low field nuclear magnetic resonance (LF-NMR), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The results showed that melanosis development was positively associated with the increase of PPO and SP activities, and the utmost melanization was observed at cephalothorax segment, following with the telson and abdomen. Besides, LF-NMR relaxometry revealed both a dramatical reduction in trapped water component T22 and a significant increase in free water component T23. Meanwhile, the histopathological findings of hepatopancreas tissue demonstrated the progressive disruption in cytoarchitecture. Along with the increase of T23 and cytoarchitecture disruption, SP and Ca²⁺ were arbitrarily disseminated in hepatopancreas tissue. In addition, a heat map analysis revealed that there was a highly positive relationship between melanosis development and the aberrant elevation of PPO and SP activities, ratio of T22 components, cytoarchitecture disruption level, and dissemination status of SP and Ca²⁺. Altogether, the biochemical cascade events for melanosis development of L. vannamei during cold storage could be sketched out; i.e., the cytoarchitecture disruption, in combination with the driving force of free water molecule migration, was greatly favorable for aggregation of SP, Ca²⁺, and PPO zymogen, following with aberrant activation of PPO zymogen and initiation of melanization. These data provide new insights into the biochemical cascade mechanism of melanosis development in L. vannamei during cold storage and pave new ways for target controlling of melanization at initial stage.

Graphical Abstract

黑色素化是虾对虾凡纳滨对虾冷藏期间感官品质的主要下降，并导致商业价值急剧下降。到目前为止，触发和加速黑素病进展的生化级联机制仍不清楚。在此，本研究旨在监测南美白对虾黑素病的发展在4°C下冷藏5天的过程中，通过应用低场核磁共振（LF-NMR），透射电子显微镜（TEM）探索丝氨酸蛋白酶（SP）在多酚氧化酶（PPO）的酶原激活中的作用和免疫组织化学（IHC）。结果表明，黑变病的发展与PPO和SP活性的增加呈正相关，并且在头胸节段观察到最大的黑化，其次是telson和腹部。此外，LF-NMR弛豫测定法揭示了截留的水组分T22的急剧减少和游离水组分T23的显着增加。同时，肝胰腺组织的组织病理学发现表明细胞结构的进行性破坏。随着T23和细胞结构破坏，SP和Ca的增加²⁺被任意散布在肝胰腺组织中。此外，热图分析表明，黑变病的发展与PPO和SP活性异常升高，T22组分比例，细胞结构破坏水平以及SP和Ca ^2+的传播状态之间存在高度正相关。总而言之，可以勾勒出冷藏过程中南美白对虾黑变病发展的生化级联事件。即细胞结构的破坏，结合水分子自由迁移的驱动力，对SP，Ca ^2+的聚集非常有利和PPO酶原，随后PPO酶原异常激活并开始黑色素化。这些数据为冷藏南美白对虾黑素病发展的生化级联机制提供了新的见识，并为初期控制黑素化目标铺平了新途径。

图形概要