富铬酵母促 GLP-1 分泌的活性肽筛选与鉴定

胰高血糖素样肽-1(Glucagon-like peptide-1, GLP-1)是一种主要由肠内分泌L-细胞分泌的具有降血糖作用的内源激素。为了从富铬酵母蛋白中筛选和鉴定出促GLP-1分泌的活性肽，文章以促GLP-1分泌活性为指导，对富铬酵母蛋白进行模拟胃肠液消化降解，采用超滤和凝胶渗透色谱法对抗消化活性肽进行分离纯化，并采用液相色谱-质谱联用仪(liquid chromatograph-mass spectrometer, LC-MS)鉴定活性肽的氨基酸序列；基于筛选出的活性肽氨基酸序列，采用固相合成法人工合成活性肽，用ELISA试剂盒测定活性肽促NCI-H716细胞和小鼠空肠分泌GLP-1的能力，用$\mathrm{Ca^{2+}}$ 试剂盒测定活性肽对胞内 $\mathrm{Ca^{2+}}$ 水平的影响。结果显示：富铬酵母蛋白酶解物具有促GLP-1分泌的肽序列，经分离纯化和结构鉴定筛选得到一条促GLP-1高效分泌的八肽(P-II)，其氨基酸序列为PSIVGRPR，分子量为 $881.04\mathrm{Da}$ ；细胞和小鼠空肠原位给药实验证明P-II可以显著促进肠内分泌L-细胞分泌GLP-1，该作用可能与其促进胞内 $\mathrm{Ca^{2+}}$ 水平升高有关。研究结果表明，富铬酵母蛋白含有促GLP-1分泌的活性肽，为富铬酵母在糖尿病防治中的应用开发提供了理论依据。

Glucagon-like peptide-1(GLP-1) is an endogenous hormone which is mainly secreted by intestinal endocrine L-cells and has the effect of lowering blood glucose. To screen and identify active peptides promoting GLP-1 secretion from chromium-enriched yeast proteins, the chromium-enriched yeast proteins were digested with simulated gastric and intestinal fluid. Under the guidance of promoting GLP-1 secretion activity, the anti-digestive active peptides were isolated and purified by ultrafiltration and gel chromatography, and their amino acid sequences were identified by liquid chromatograph-mass spectrometer(LC-MS). On this basis, active peptides were artificially synthesized by solid phase method. GLP-1 content in NCI-H716 cells and mouse ileum was determined by ELISA, and intracellular$\mathrm{Ca^{2+}}$ content was determined by $\mathrm{Ca^{2+}}$ kit. The results showed that the protein hydrolysates of chromium-enriched yeast contained peptide sequences that promoted GLP-1 secretion. An octapeptide, P-II, was obtained by isolation, purification, and structural identification. The amino acid sequence of P-II was PSIVGRPR and its molecular weight was 881.04 Da. In situ administration of cells and mouse jejunum showed that P-II could significantly promote the secretion of GLP-1 from intestinal endocrine L-cells, which might be related to the increase of intracellular $\mathrm{Ca^{2+}}$ level. These studies indicate that chromium-enriched yeast proteins contain active peptides that promote GLP-1 secretion, which provides theoretical basis for the application and development of chromium-enriched yeast in the prevention and treatment of diabetes.