厚味（Kokumi）γ-谷氨酰肽增鲜作用的分子机制研究

Gamma-glutamyl peptides have several taste characteristics, namely, they mainly exhibit an astringent taste in the aqueous, and a few exhibit also umami or bitter taste. When added to foods, γ-glutamyl peptides can impart the desirable kokumi taste attributes of thickness, mouthfulness and continuity. Some of the so-called “kokumi” γ-glutamyl peptides like γ-[Glu]n-Val/Phe/Met, γ-Glu-Val-Gly and γ-Glu-Cys-Gly can also enhance the perceived intensity of the umami taste of MSG and chicken broth, but the umami-enhancing mechanism is still unclear. This study will focus on the molecular mechanism of the umami-enhancing effect of γ-glutamyl peptide. Molecular docking and simulation are used to analyze the optimal conformation, binding site and molecular dynamic process of the combination among γ-glutamyl peptides, taste receptor T1R1/T1R3 and umami substances, as well as the effect of γ-glutamyl peptides on the combination of umami receptor and umami receptor will be obtained. The activation of γ-glutamyl peptides on umami receptors will be demonstrated via cell test, and the binding sites of γ-glutamyl peptides and umami receptors will be analyzed by site-directed mutagenesis of umami receptors. Overall, this study attempts to obtain the umami-enhancing molecular mechanism of the so-called “kokumi” γ-glutamyl peptides, to provide a research method and theoretical basis for studying the umami-enhancing mechanism of such taste substances which have no umami but can enhance the umami taste, to improve the taste characteristic description of γ-glutamyl peptides and obtain the γ-glutamyl peptides that have the kokumi and umami-enhancing effect.

γ-谷氨酰肽具有多变的呈味特性，即它们在水溶液中主要呈酸涩味；添加到食物中可赋予食物浓厚感、满口味和持续性等厚味（kokumi）味感；部分厚味γ-谷氨酰肽如γ-[Glu]n-Val/Phe/Met、γ-Glu-Val-Gly、γ-Glu-Cys-Gly还可增加味精和鸡汤的鲜味，但增鲜机制尚不明确。本项目试图借助分子模拟技术和细胞实验，解析γ-谷氨酰肽增鲜作用的分子机制。通过分子模拟找出γ-谷氨酰肽与鲜味受体-鲜味物质复合体结合的最佳构象，与鲜味受体结合的位点、键长、键能和动力学过程。通过细胞实验确定γ-谷氨酰肽对鲜味受体的激活作用，获得γ-谷氨酰肽与鲜味受体的结合位点。加以感官实验的验证，为研究不具有鲜味却具有增鲜作用的呈味物质的增鲜机制提供研究方法和理论基础。同时丰富γ-谷氨酰肽呈味特性的描述，获得既具有厚味特性又具有显著增鲜作用的γ-谷氨酰肽。

厚味γ-谷氨酰肽被认为可增加味精和鸡汤的鲜味，但增鲜机制尚不明确。本项目采用了感官分析、分子模拟技术和细胞实验验证，解析了厚味γ-谷氨酰肽的增鲜作用及分子机制。结果显示，①首先通过人体感官和电子舌测定了24种常见厚味γ-谷氨酰肽的增鲜效果，其中人体感官评价的鲜味评分增加范围为0.39 ~ 0.72（p<0.05），电子舌评价为0.81 ~ 3.05（p<0.05），表明厚味γ-谷氨酰肽具有增鲜效果。②为了解析其增鲜机理，构建了一种新的受体—鲜味受体T1R1、T1R3与味精（MSG）的受体复合物（T1R1-MSG和T1R3-MSG）。γ-谷氨酰肽与这两种受体复合物的分子对接得分分别为5.569 2-8.857 8和6.1583-9.4253-4，说明这些多肽对T1R3具有更强的亲和力。T1R1的24个氨基酸残基以及T1R3的28个氨基酸残基作为结合氨基酸位点，分别参与配体与受体复合物的相互作用。通过分子动力学模拟分析进一步比较了三种厚味γ-谷氨酰肽（γ-E-γ-EV、γ-EL、γ-EV）在增鲜作用上的差异。其中范德华力(ΔEvdw)是配体和受体之间总结合自由能的主要贡献者，γ-E-γ-EV/γ-EL-/γ-EV-T1R3体系的总结合自由能(ΔGbind)分别为-37.50±2.87、-28.79±3.29和-28.31±2.56 kcal/mol，γ-EL和γ-EV在受体-配体相互作用能上没有差异，但明显低于γ-E-γ-EV。在T1R3分子中发现了多达9、8和6个氨基酸残基，均包含了Glu-301, Ala-302, Thr-305和Ser-306四个氨基酸残基，分别在受体-配体相互作用中发挥了关键作用。③细胞实验验证了这三条γ-谷氨酰肽的增鲜作用生理机制。结果显示它们与味精混合可协同提高细胞内钙释放量(108.47±7.89%~152.33±26.26%)，增加T1R1/T1R3蛋白表达。T1R3的氨基酸残基位点V277, S147和D190对于γEγEV与T1R3的结合至关重要。.通过本项目的开展明确了厚味γ-谷氨酰肽的增鲜作用及作用机制，同时为研究不具有鲜味却具有增鲜作用的呈味物质的增鲜机制提供研究方法和理论基础，丰富了γ-谷氨酰肽呈味特性的描述，获得既具有厚味特性又具有显著增鲜作用的γ-谷氨酰肽。