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采用超声协同稀碱对猪皮实施预处理后,酶解30 min制得的高血管紧张素转化酶(Angiotensin converting enzyme,ACE)抑制活性水解液为原料,采用Sephadex G-15、半制备高效液相色谱分离纯化其中胶原ACE抑制肽并对其序列进行鉴定。结果表明,Sephadex G-15分离胶原ACE抑制肽的最佳分离条件为:样品浓度100 mg/m L;上样量2 m L;流速2 m L/min;洗脱剂为蒸馏水;层析柱为1 m×10 mm的层析柱。在该分离条件下,混合肽被分成AP-I、AP-II两个组分,IC50值分别为19.50、1.01 mg/m L。对抑制活性较强的AP-II进一步采用半制备高效液相色谱进行分离,得到8个组分峰,其中峰2、峰5和峰6的IC50值最小,分别为0.73、0.44和0.4 mg/m L。结合IC50值的大小及半制备收集到样品的量,对峰2和峰6采用LC-MS/MS进行序列分析。结果显示峰2的多肽序列为QGPPGPAGPR(P为Hyp),峰6的序列为AGPPGPPGPA,这两个序列位于胶原蛋白α1链中526?535、730?739位。
After the pigskin was pretreated by ultrasonic synergistic dilute alkali, the ACE inhibitory active hydrolyzate prepared by enzymolysis for 30 min was used as raw material. Sephadex G-15, The chromatographic separation and purification of collagen ACE inhibitory peptides and their sequences were identified. The results showed that the best separation conditions of Sephadex G-15 isolating collagen ACE inhibitory peptides were as follows: the sample concentration was 100 mg / mL, the sample volume was 2 mL, the flow rate was 2 mL / min, the eluent was distilled water, A 1 m × 10 mm column. Under the conditions of separation, the mixed peptide was divided into AP-I, AP-II two components, IC50 values were 19.50,1.01 mg / m L. The more inhibitory AP-II was further separated by semi-preparative high performance liquid chromatography to obtain eight component peaks, wherein the IC50 values of peak 2, peak 5 and peak 6 were the smallest, which were 0.73, 0.44 and 0.4 mg / m L. According to the size of IC50 and the amount of sample collected by semi-preparation, the sequence analysis of peak 2 and peak 6 was performed by LC-MS / MS. The results showed that the polypeptide sequence of peak 2 was QGPPGPAGPR (P is Hyp), the sequence of peak 6 was AGPPGPPGPA, and the two sequences were located at 526-535,730-739 of the collagen α1 chain.