高温高压即食海参体壁非酶自降解途径及其拮抗机制

The enzymes and microorganisms of the ready-to-eat sea cucumber treated by high pressure steam (HSC) are inactivated, but it is easy to appear serious non-enzymatic self-degradation phenomenon after a period of storage time. It restricts the development of the ready-to-eat sea cucumber industry. However, there are few reports on the pathway of non-enzymatic self-degradation and the intervention mechanism of degradation. Basing on non-enzymatic degradation problem of the HSC body wall in this study, and the regulation of non-enzymatic degradation and changes of main chemical force will be analyzed during storage. Taking the non- enzymatic cleavage site of sea cucumber collagen peptide chain as breakthrough point innovatively, the characteristic peptide model is designed. The pathway of HSC non-enzymatic self-degradation can be clarified by calculating energy changes in the molecular self-cleavage process by molecular simulation, analyzing reaction rate limiting steps, analyzing the configuration of fracture fragment, and utilizing the information obtained by isotope labeling, tandem mass spectrometry, and other test technology. Phenol antioxidant gallic acid is employed for cross-linking HSC innovatively, and the gel properties, microstructure characteristics and thermal stability are systematically evaluated. Furthermore, the effect of molecular cross-linking on the stability of HSC body wall is explored by the crystalline structure, secondary structure, hydrogen bond, hydrophobic bond and side chain groups. Finally, the intervention mechanism of molecular cross-linking on HSC non-enzymatic self-degradation is clarified by comparing the changes of non- enzymatic cleavage site, fracture mode, and fracture pathway. It can provide the scientific basis for processing of sea cucumber.

尽管高温高压即食海参（HSC）中微生物和内源酶已失去活性，但其贮藏中会出现严重的非酶自降解现象，困扰着即食海参产业的发展，而目前关于HSC非酶自降解途径及其干预机制却鲜有报道。本课题以HSC体壁非酶劣化问题为出发点，分析其在贮藏中非酶劣化规律与主要化学作用力变化趋势，独具创新地以“海参胶原肽链的非酶断裂位点”为突破点，设计特征肽段模型，借助分子模拟手段计算自裂解过程能量变化、分析决速步骤、解析断裂构型，同时结合同位素标记、串联质谱等技术阐明HSC肽链的非酶裂解途径。创新地采用酚类抗氧化剂没食子酸交联HSC，并系统评价其凝胶性能、微观结构特征、热稳定特性，进一步通过交联前后HSC晶相结构、二级结构、氢键、疏水键以及侧链基团的变化等解答分子交联稳定HSC体壁凝胶的模式；通过交联前后HSC肽链断裂位点、断裂方式以及裂解途径的变化，阐明分子交联对HSC非酶自降解的拮抗机理，为其加工提供科学依据。

高温高压即食海参（HSC）的非酶自降解问题是海参加工业的瓶颈难题。本课题解析了HSC在贮藏中非酶劣化规律，以“非酶断裂位点”为突破点，设计特征肽段模型，借助量子化学手段计算自裂解过程能量变化，分析决速步骤，解析断裂构型，同时结合串联质谱等技术阐明HSC的非酶裂解途径。采用酚类分子交联HSC，评价了其微观结构、热稳性能变化，基于交联前后HSC二级结构、次级键等的变化，解答分子交联稳定HSC模式，阐明了分子交联对HSC非酶自降解的拮抗机理。取得的重要成果与关键数据如下：HSC的硬度和咀嚼性的半数衰减周期（T50）均随着贮藏温度、水分含量降低而显著性增加。建立了硬度非酶劣化模型lnA=-0.0625t+1.6157；随贮藏时间延长，胶原纤维断裂加剧，自由水比率增高；氢键是稳定HSC的主要次级键。HSC非酶自降解过程中游离羟脯氨酸(FHYP)与游离氨态氮(FAN)显著增加，共价键发生了自断裂。利用质谱非标定量法筛析了HSC肽链断裂位点为G、Q、N、D、L、E、A、R、S、Y、K、H，同时利用Edman降解法识别胶原肽链断裂位点为S、D、H、E、V。进一步设计了特征肽段模型CH3CO-N-A-NHCH3等，基于B3LYP/6-31G(d)等确定了每步裂解中反应物、过渡态、产物的自由能，查明了天冬酰胺侧链环化成五元环为整个反应的决速步骤，相对于A、I、G、Q，N与L、D、V形成的二肽模型更容易水解断裂。采用没食子酸（GA）、绿原酸（CA）等交联HSC，ε-氨基与硬度呈现显著负相关，贮藏期间的HSC微观结构特征、水分迁移均显著改善，随着交联度增加，热稳定性增强。解析了交联HSC热降解动力学，GA与CA交联HSC活化能分别为 302.07-356.28 kJ/mol与254.99-382.13 kJ/mol。交联干预了HSC的无规则卷曲比例增加，拮抗了FHYP与FAN增加，减缓了HSC降解趋势，改变了肽链断裂位点，但主要次级键仍是氢键。基于量子化学与合成肽验证，查明了交联特征肽段CH3CO-N-G-NHCH3等在自降解过程中能量势垒的升高，阐明了交联拮抗机理，为即食海参加工提供了理论指导。