养殖东方鲀呈味肽形成机制及其构效研究

Takifugu is popular among consumers for its delicious taste, and taste-active peptides are the important compounds contributed to its palatable taste such as "mellowness" and "tenderness". So far no report was found about taste peptide formation mechanism and structure-activity relationship of Takifugu. Cultured Takifugu obscurus and Takifugu rubripes under different processings including chilling, heat processing and enzyme treatment are as samples. Application of modern separation and purification technology including membrane separation, gel filtration chromatography and semi-preparative reversed-phase high performance liquid chromatography to obtain high purity of the oligopeptides. Mass spectrometry, protein N-terminal sequencing techniques, taste dilution analysis, the group deduction and addition sensory analysis combine with the electronic tongue are used to identify the taste-active peptide contributed to the puffer characteristics flavor. We would take the identified peptide as model and change the main taste amino acid to ascertain its structure-activity relationship by chemical synthesis, computer software simulation and sensory analysis combine with the electronic tongue. We would compare the structure, taste characteristics and the change tendency of flavor peptides from puffer fish under different processings to determine the effect of the endogenous enzyme, exogenous enzymes and thermal processing on the flavor peptides. Nucleotide and protein database was queried to identify the taste peptide protein sequence corresponding to the protein and gene so as to track the origin, which would explore the possible formation mechanism of taste-active peptides. It has important scientific and practical significance to processing, utilization and development of the simulation seasonings of the Takifugu to investigate the key flavor peptides formation mechanism and structure-activity relationship.

河鲀滋味鲜美独特，深受消费者喜爱。呈味肽是构成河鲀鲜美浓郁、圆润香滑等味感的重要风味物质之一，目前国内外对河鲀呈味肽形成机制及其构效研究未见报道。本项目拟以养殖暗纹东方鲀和红鳍东方鲀为对象，对冷藏、热加工及酶处理的河鲀肌肉进行研究。应用膜分离、凝胶过滤色谱以及半制备反相高效液相色谱从河鲀提取物中获得高纯度小肽；利用质谱及蛋白质N端测序技术和感官评定结合电子舌鉴定河鲀特征滋味肽。以鉴定的肽链为模型，改变主要呈味氨基酸，通过化学合成、电脑软件模拟以及电子舌、感官评定探明其构效关系。通过分析不同处理河鲀中呈味肽的结构、呈味特性及其变化趋势，确定内源酶、外源酶以及热加工对呈味肽的作用；查询核酸蛋白序列数据库识别鉴定呈味肽的相关蛋白质序列，分析呈味肽所对应的蛋白质及基因，对其进行溯源，以期探究呈味肽的可能形成机制。该项目的开展将为揭示河鲀美味奥秘、河鲀加工利用及其调味料开发提供理论基础和技术支持。

呈味肽是构成河鲀鲜美浓郁、圆润香滑等味感的重要风味物质之一。本项目以养殖暗纹东方鲀和红鳍东方鲀为对象，对冷藏、热加工及酶处理的河鲀肌肉应用膜分离、凝胶过滤色谱以及半制备反相高效液相色谱从河鲀提取物中获得高纯度小肽；利用质谱及蛋白质N端测序技术和感官评定结合电子舌鉴定河鲀特征滋味肽。研究结果表明共新发现鲜味浓郁的呈味肽10条，分别是Glu-His-Ala-Met-Leu-Asn（714.2917 Da）、Pro-Gly-Gly-Val-Arg-Asn-Gly（656.0368Da）、Cys-Ala-Leu-Thr-Pro（503.9253Da）、Arg-Pro-Leu-Gly-Asn-Cys（659.7747Da）、Pro-Val-Ala-Arg-Met*-Cys-Arg（810.9046Da，Met*为氧化型甲硫氨酸）和Thr-Leu-Arg-Arg-Cys-Met*（794.9372 Da）、Val-Arg-Ser-Tyr（534.24 Da）、Tyr-Lys-Cys-Lys- Asp-Gly-Asp-Leu-Arg（1079.56 Da）、Leu-Tyr-Glu-Arg（580.25 Da）、和Lys-Gly-Arg-Tyr-Glu-Arg（808.37 Da）。通过模拟红鳍东方鲀肌肉鲜味轮廓以研究呈味肽的呈味特性，结果发现添加化学合成呈味肽的人工复配液能较好地模拟生鲜红鳍东方鲀肌肉天然的鲜味，且呈味肽的存在对其他滋味物质鲜味的呈现具有重要的协同作用，能明显地提升整体溶液的鲜味强度。探究了内源性组织蛋白在暗纹东方鲀肌肉呈味肽形成过程中的作用，发现冷藏3天后其鲜味特性最强；内源性组织蛋白酶B、L和H酶的活力越强，鲜味值越高；组织蛋白酶B与L降解蛋白使得肽含量积累，并且有一部分还在氨肽酶作用下降解为氨基酸，所有游离氨基酸的总量也不断增加；组织蛋白酶H对暗纹东方鲀呈味肽形成的作用较小。通过分子模拟手段构建了鲜味六肽空间结构，建立了鲜味六肽间静电场及立体场差异和三维定量构效关系模型，部分解释了肽结构与鲜味强度之间的关系，为后续改造已知鲜味肽和设计更鲜的呈味肽提供了基础。本项目不仅丰富了鲜味感知理论，而且还为揭示河鲀美味奥秘、河鲀加工利用以及高品质呈味基料提供了理论基础和技术指导，具有较为重要的理论价值和现实意义。