麦胚降血糖肽的分离纯化及鉴定

建立麦胚降血糖肽的分离纯化方法及鉴定活性多肽的结构组成。采用胰蛋白酶酶解麦胚蛋白,得到降血糖多肽,并进行降血糖动物实验。超滤获得高活性组分,离子交换吸附实验选择最佳的树脂,并对麦胚降血糖肽进行离子交换吸附分离,SephadexG-25和SephadexG-15分离,再经过反相高效液相色谱(reverse phase-high performance liquid chromatography,RP-HPLC)分离高活性成分。最后采用高效液相色谱-电喷雾-质谱(HPLC-electrospray ionization-massspectrometry,HPLC-ESI-MS)鉴定活性多肽的结构组成。结果显示酶解麦胚蛋白对α-葡萄糖苷酶的抑制性IC_(50)为10.98 mg/mL,能够缓解糖尿病小鼠的症状。超滤获得分子质量小于5 kDa的高活性组分,IC50为1.60 mg/mL。732型阳离子交换树脂的分离效果最好,2.5%氨水的洗脱率为98.13%,通过动态离子交换吸附分离后,活性显著提高,IC_(50)为0.30 mg/mL。通过SephadexG-25和SephadexG-15分离得到高活性组分,经RP-HPLC分离得到8个组分(峰),其中1号峰的活性和含量都最高,IC_(50)为0.098 mg/mL。HPLC-ESI-MS结果显示m/z 274.45的丰度比较高,经离子碎片拼接,共有7种多肽,其中二肽有1种,三肽有6种。本研究对于开发具有降血糖功效的新型功能性食品有一定的作用。

Isolation and Structural Identification of Hypoglycemic Peptides from Wheat Germ Protein

The objective of the present study was to purify and structurally identify hypoglycemic peptides from wheat germ protein. The protein was hydrolyzed with trypsin to obtain the hypoglycemic peptides, and the hypoglycemic effect was evaluated by animal test. The active peptides were obtained by ultrafiltration. The optimum resin was selected for purification of the peptides by ion exchange adsorption, and further purification was carried out by sequential column chromatography on SephadexG-25 and SephadexG-15 before separation by reverse phase-high performance liquid chromatography (RP-HPLC). Finally, the structures of the purified peptides were identified by HPLC-electrospray ionization-mass spectrometry (HPLC-ESI-MS). The results showed that the enzymatic hydrolysate had α-glucosidase inhibitory activity with an IC50 of 10.98 mg/mL, and could relieve symptoms in diabetic mice. The highly active peptides with molecular mass less than 5 kDa were obtained by ultrafiltration, and their IC50 was 1.60 mg/mL. 732 cation exchange resin showed the best separation efficiency, and the elution percentage with 2.5% ammonia was 98.13%. After ion exchange adsorption, the hypoglycemic activity was significantly improved, yielding an IC50 of 0.30 mg/mL. Eight peaks were obtained after purification by RP-HPLC and among these the amount and activity of peak 1, with an IC50 of 0.098 mg/mL, were both highest. HPLC-ESI-MS analysis showed that the abundance at m/z 274.45 was high and that 7 peptides including 1 dipeptide and 6 tripeptides were obtained by ion fragment re-assembling. The findings obtained in this work are of significant importance for the development of novel functional foods with hypoglycemic efficacy.