养殖东方鲀呈味肽形成机制研究

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 and its formation mechanism 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 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 its key flavor peptides and their formation mechanism.

河鲀滋味鲜美深受消费者喜爱，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，目前国内外对河鲀呈味肽及其形成机制未见报道。本项目拟以养殖暗纹东方鲀和红鳍东方鲀为对象，对生鲜、冷藏、热加工及酶处理的河鲀肌肉进行研究。应用现代分离纯化技术膜分离、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的寡肽；利用质谱及蛋白质N端测序技术、滋味稀释分析方法、组别减除、添加感官评定结合电子舌鉴定河鲀特征滋味肽。通过分析比较不同处理河鲀中呈味肽的结构、呈味特性以及变化趋势，确定相关内源酶、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、河鲀加工利用及仿真调味料的开发提供理论基础和技术支持。

本项目以养殖暗纹东方鲀(Takifugu obscurus)为研究对象，鉴定了经热加工以及冷藏处理提取的暗纹东方鲀肌肉中的未知呈味肽（Takifugu obscurus flavor peptide, PFFP），探究了其滋味特性，并研究了暗纹东方鲀肌肉中的已知滋味相关肽如肌肽和谷胱甘肽。该项目培养2位研究生，发表及录用学术论文各1篇，申请专利1项，按计划完成了项目的各项指标。主要研究结果如下：.（1）热加工以及冷藏处理暗纹东方鲀肌肉中的呈味肽分离鉴定。采用超滤初步分离出提取液中分子量小于3000Da的肽类，分离液经Sephadex G-15凝胶层析纯化得到5个多肽组分（P1、P2、P3、P4 和P5）。感官评定结合电子舌分析测定表明5个多肽组分中P2组分滋味特性最强，氨基酸分析其肽含量也最高为0.19 mg/mL。采用反相高效液相色谱将P2组分进一步分离成了3个多肽组分P2a、P2b 和 P2c。经电子舌测定出P2b滋味特性最强，且具有鲜味和甜味。利用MALDI-TOF/TOF MS/MS鉴定得到P2b 的氨基酸序列为Tyr-Gly-Gly- Thr-Pro-Pro- Phe-Val (836.4 Da)。在冷藏处理的样品中鉴定得到六肽Pro-Val-Ala-Arg-Met*-Cys-Arg（810.9046Da），具有显著的浓厚感。.（2）应用电子舌技术探究了热加工处理鉴定的暗纹东方鲀呈味肽的滋味特性。人工合成的PFFP的鲜味和甜味比天然提取的PFFP分别高2个和3个分数等级。100ºC加热30 min后能增加PFFP的鲜味、甜味；增加pH4.0 PFFP的鲜味，降低其甜味。在pH4.0~pH9.0范围内，改变PFFP 的pH值，鲜味降低，PFFP-100ºC鲜味最强；增加PFFP的pH值，其甜味降低。MSG、NaCl和MSG+NaCl都具有增加PFFP的鲜味，降低甜味的作用，其中NaCl可将PFFP的鲜味强度值从2.19提升至7.45，与NaCl+MSG+ PFFP的鲜味强度值7.46相当。.（3） 采用薄层色谱与高效液相色谱联用技术鉴定了暗纹东方鲀肌肉中的肌肽和谷胱甘肽，并利用HPLC进行定量分析。结果表明：暗纹东方鲀肌肉中肌肽含量约0.213 µg/g (鲜重)，还原性谷胱甘肽含量约0.211 µg/g (鲜重)。