鲜味肽呈鲜效果的基因表达验证及其呈鲜机理的研究

Umami peptide is a recent discovered new umami agent, its flavoring effect is widely disputed. Study found umami peptides separated and purified from enzymatic hydrolysate were concluded having no umami taste as their flavor was verified with synthesized peptides. The chemical synthesis of peptides may lead to the change of amino acids’ conformation and peptide’s spatial structure, thus affecting the flavor of synthetic peptides. In order to further verify the taste of umami peptide and clarify its flavoring mechanism, a controversial umami octapeptide will be taken as subject, according to the genetic information of the peptide’s amino acid sequence, genetic engineering technique will be used to express the peptide in Pichia pastoris to obtain the peptide with natural structure, and then using the natural peptide to verify the taste of the octapeptide. Based on the investigation, the native peptide will be modified by replacing the L- amino acids with D, knocking out some amino acids and combining other chemical groups in the residue of amino acids. The spatial structure change of the native peptide before and after modification will be analyzed with circular dichroism analysis. The relationship between the special structure modification and its flavor change will be analyzed so as to investigate the key factors of affecting the taste of umami peptide and clarify the peptide’s flavoring mechanism. This will not only provide new evidence to solve the controversy of the taste of umami peptide, supply raw materials for the study of umami peptide receptors, but also found a theoretical basis for the industrial production of umami peptides, enhancing the flavor of artificial meat and other applications.

鲜味肽是近年来新发现的增鲜剂，国内外对其呈鲜效果存在争议。从酶解产物中分离纯化的鲜味肽，当用化学合成该肽法验证其呈鲜效果时出现矛盾的结果。化学合成肽时可能导致氨基酸构象和肽空间结构的改变，因而影响了合成肽的风味。为了进一步明确鲜味肽的呈鲜效果及其呈鲜机理，本课题拟以争议较大的鲜味八肽为研究对象，根据该肽的氨基酸序列对应的基因信息，采用基因工程技术将其在毕赤酵母中重组、表达，以获得天然结构肽，进而验证其呈鲜效果。在此基础上对该天然肽中的氨基酸进行构象替换、敲除及残基修饰，利用圆二色谱分析天然肽修饰前后的空间结构，通过天然肽的空间结构改变与其风味变化的相互关系，探明影响鲜味肽呈味的关键因素，以揭示鲜味肽的呈鲜机理。这不仅可为解决鲜味肽呈鲜效果的争议提供新证据，为鲜味肽受体的研究提供原料，而且可为工业化生产鲜味肽、提高人造肉的鲜味等应用提供理论依据。

鲜味肽是近年来发现的新型增鲜剂，在国内外的研究报道中多数鲜味肽的呈鲜效果存在一定的争议。当用化学法合成法验证从酶解产物中分离纯化的相同一级结构鲜味肽呈味效果时，其结果有一定的矛盾。在化学合成肽的过程中可能导致氨基酸构象和肽空间结构发生改变，从而影响合成肽的风味。为了进一步明确鲜味肽的呈鲜效果及其呈鲜机理，本课题拟以争议较大的鲜味八肽（Lys-Gly-Asp -Glu-Glu-Ser-Leu-Ala, LGAGGSLA）为研究对象，根据该肽的氨基酸序列对应的基因信息，采用基因工程技术将其在大肠杆菌中重组、表达，以获得天然结构肽，为进一步验证其呈鲜效果提供原料。.根据LGAGGSLA的氨基酸序列和宿主大肠杆菌密码子表达的偏爱性，设计了LGAGGSLA的融合蛋白核酸序列，定向克隆到质粒pET-32a（+）上形成表达载体。经检测菌落PCR阳性克隆，重组质粒DNA测序吻合，诱导表达的融合蛋白丙烯酰胺电泳检测大小吻合，得出所构建LGAGGSLA的表达载体符合预期，形成的工程菌能成功表达融合蛋白。在此基础上，对工程菌的诱导条件进行优化，得出OD600值为0.5，IPTG浓度为0.6mM，诱导温度28.75℃，诱导时间5h时，工程菌融合蛋白诱导表达量最高。通过对工程菌表达蛋白酶切工艺进行优化，得出温度为24℃、时间15h、酶浓度2μL/mL时，融合蛋白的酶切率最高，酶切率达93.4%。酶切液先后经亲和RPC反向层析、阳离子交换层析纯化后，经HPLC验证，与人工合成的标准品的保留时间基本一致，且质谱对目标肽测序结果吻合。.通过本项目实施，形成了以教授为带头人、青年博士为骨干的鲜味肽研究团队1个，培养教授1人、硕士研究生2人，发表学术论文4篇，其中SCI论文3篇，受理发明专利2项，撰写研究报告1份。