羊乳清蛋白ACE及DPP-IV抑制肽的分离纯化与鉴定

Purification and identification of angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory peptides from goat whey protein hydrolysates

关键词:  羊乳清蛋白  ACE抑制活性  DPP-IV抑制活性  双重活性多肽

Key words:  goat whey protein  angiotensin-converting enzyme  dipeptidyl peptidase IV  dual bioactive peptide

摘要:

使用中性蛋白酶、碱性蛋白酶、胰蛋白酶、风味蛋白酶和木瓜蛋白酶对羊乳清蛋白进行水解，分别在不同时间取样，体外测定其ACE抑制活性和DPP-IV抑制活性。通过中空纤维膜超滤、凝胶过滤层析和反向高效液相色谱（RP-HPLC）的手段对酶解产物进行逐步分离和纯化，评价各级分离产物的生物活性；通过液质联用（LC-MS/MS）的方法鉴定多肽的氨基酸组成，根据质谱结果合成多肽。结果表明：中性蛋白酶水解物具有一定的生物活性，双重活性多肽Pro-Pro-Ala-Ser-Glu-Val-Val-Lys-Pro对ACE和DPP-IV均有抑制作用，其IC₅₀值分别是573.32±4.63和2 065.35±22.30 μmol/L。Ile-Pro-Ala-Val-Phe-Lys-Ile-Asp具有较好的DPP-IV抑制活性，其IC₅₀值为964.14±4.09 μmol/L。羊乳清蛋白水解物可以作为食源性材料用于管理伴有高血压的Ⅱ型糖尿病患者的饮食。

Abstract:

Goat whey protein was hydrolyzed with neutrase, alcalase, trypsin, flavourzyme and papain. The hydrolysates were sampled at different time points. ACE and DPP-IV inhibitory activities were measured at the concentration of 5 mg/mL. The neutrase-generated hydrolysate was isolated by ultrafiltration, gel filtration chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC) and then identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The peptides were synthesized and assayed for bioactivities. The results showed that the hydrolysate treated with neutrase showed certain bioactivities. The dual bioactive peptide Pro-Pro-Ala-Ser-Glu-Val-Val-Lys-Pro showed inhibition against ACE and DPP-IV with IC₅₀ values of 573.32±4.63 and 2 065.35±22.30 μ mol/L, respectively. Ile-Pro-Ala-Val-Phe-Lys-Ile-Asp had better DPP-IV inhibitory activity with an IC₅₀ value of 964.14±4.09 μ mol/L. In conclusion, the goat whey protein hydrolysates can be used as a food-derived material for managing the diet of type 2 diabetes mellitus patients with hypertension.