海参低温嫩化过程中体壁胶原组织的微观结构变化及机理

Thermal ripening is key processing step in production of ready-to-eat sea cucumber. Our research team has developed a low-temperature tenderization technology. After the tenderization processing, sea cucumber was rapidly processed by a high-temperature ripening step, and its tenderness was better than the one without low-temperature tenderization processing. Tenderization is a common technology in meat processing. It was reported that the nature for tenderization of meat is a limited hydrolysis of muscle tissue by endogenous proteases. In contrast with meat, sea cucumber body wall is mainly mutable connective tissue (MCT). Our previous studies indicated that the activity of cathepsin L in sea cucumber body wall was activated during low-temperature tenderization processing. Meanwhile, the periodic cross striation of fibril disappeared. In such cases, our research team speculated that the nature of low-temperature tenderization of sea cucumber body wall is limited degradation of MCT caused by endogenous proteases. The main purpose of this project is to reveal the mechanism of low-temperature tenderization of sea cucumber body wall though investigation of microstructural changes of MCT and the roles of endogenous proteases in the changes. To achieve the goal, our team intend to study the microstructural changes of MCT of sea cucumber body wall during low-temperature tenderization; our team intend to study the soluble components of MCT of sea cucumber body wall during low-temperature tenderization; our team intend to study the effects of inhibition of endogenous proteases on low-temperature tenderization of sea cucumber body wall by in situ perfusion technology. The present study also can provide evidence for low-temperature tenderization of other sea animals.

加热熟化是即食海参加工的关键步骤。申请人所在课题组建立了低温嫩化技术，采用此技术处理的海参熟化后体壁嫩度好于直接熟化的海参。嫩化技术是肉制品加工的常用技术，其机理是内源蛋白酶对肌肉组织的有限降解。与肉制品不同，海参体壁主要由易变胶原组织（mutable connective tissue, MCT）构成。课题组前期研究表明，海参体壁组织蛋白酶L在低温嫩化过程中活性增强，同时海参胶原原纤维周期性横纹结构消失。据此课题组推测，海参体壁低温嫩化的实质是内源酶系对MCT的有限降解。本项目拟通过研究海参体壁MCT微观结构在低温嫩化过程中的变化及各内源酶系在MCT微观结构变化中发挥的作用，揭示低温嫩化机理。为了实现预期目标，课题组将研究海参经低温嫩化处理后体壁MCT微观结构变化及溶出成分变化；研究内源酶被抑制的海参经低温嫩化处理后体壁的嫩化效果。通过本研究，也可为其他海洋动物的低温嫩化技术提供依据。

如何控制嫩度是即食类海参品质的核心关键。只有明确海参嫩化过程的作用机制，才能找到更有效的嫩度调控方法。项目围绕海参体壁易变胶原组织（MCT）在低温嫩化过程中变化及内源酶在变化中发挥作用开展研究，揭示了低温嫩化机理。.1.采用光镜、组织化学染色结合扫描电镜等技术观察嫩化处理前后海参体壁微观组织结构变化。随嫩化进行，胶原纤维由紧密有序，逐渐交织，至出现凝集；胶原原纤维间蛋白聚糖的桥连结构出现断裂，纤维间开始分离，局部出现解螺旋现象。.2.采用链霉蛋白酶作用结合色谱等方法考察了低温嫩化过程中海参胶原中可溶性、酶溶性和总不溶性成分以及糖胺聚糖等含量的变化。处理后的海参体壁可溶性胶原蛋白、酶溶性胶原蛋白含量升高，不溶性胶原蛋白含量逐渐降低，验证了胶原组织结构发生降解；水溶性糖胺聚糖含量增加也间接说明蛋白聚糖桥结构破坏。.3.红外光谱发现嫩化处理促使海参粗胶原纤维（CCF）酰胺I带破坏，30min时二级结构变化明显，推测α螺旋向β折叠转变；流变仪分析发现嫩化过程对海参体壁组织G’和Tanδ等流变学特性、吡啶交联含量影响显著，30 min时G’剧烈上升表明开始向凝胶态转变；低场核磁显示了嫩化过程海参体壁组织水分相态及分布变化，弛豫时间向左迁移，自由水相对比例持续降低，结合水和不可移动水比例增加。.4.采用酶活力检测法考察嫩化过程海参内源性蛋白酶活力变化，组织蛋白酶L在45℃，15-60min和50℃，0-30min、组织蛋白酶B在45℃和50℃，15-30min、钙激活蛋白酶在40℃-45℃，30-60min内酶活较强；新鲜海参蛋白酶L位点主要集中在近皮外层，中间层及内层较少，随时间延长由外向内扩散；初步构建海参体壁胶原纤维实验模型，进一步验证了内源酶在嫩化中的作用。.综上多种手段共同论证，验证了海参低温嫩化处理本质是内源酶引发的MCT结构适度降解，但不同酶系发挥作用存在差异。本研究丰富了海参低温加工理论，为其他海洋动物低温嫩化提供依据。