为了得到新的ACE抑制肽，采用分子排阻色谱对核桃蛋白碱性蛋白酶水解物进行分离、纯化。优化了分子排阻色谱分离、纯化核桃蛋白水解物的洗脱剂、洗脱流速、上样体积和样品浓度，比较了其纯化前后ACE抑制活性变化、分析了不同组分RP–HPLC图谱及其氨基酸组成。结果表明，以Sephadex G–15为分离介质的色谱优化条件为：超纯水为洗脱剂、洗脱流速0.4 mL/min、上样体积1.5 mL、样品浓度150 mg/mL。在此条件下，经过分子排阻色谱纯化后，得到4个主要组分，其中活性最高组分的ACE抑制率达90.35±0.25%，其IC50值为0.158 mg/mL。经过RP–HPLC进一步分离，分子排阻色谱纯化后高活性组分保留时间主要集中于10 min、13 min、17 min和21 min，并且其疏水性氨基酸的含量得到显著富集，其中芳香族氨基酸Tyr、Phe的含量分别达10.46%和20.15%。%In order to obtain the novel angiotensin I–converting enzyme(ACE)inhibitory peptides, walnut protein hydrolysates prepared with Alcalase 2.4 L were isolated by size exclusion chromatography. Firstly,the operation parameters of size exclusion chromatography such as the eluent,the flow rate and the sample concentration were investigated. Furthermore,the ACE inhibitory activity of different parts were assessed before and after the purification. Additionally,the RP–HPLC map and amino acid composition of the ACE inhibitory peptides were determined. The results showed that the distilled water was the best eluent,the elution rate was 0.4 mL/min,the sampling volume was 1.5 mL,the sampling concentration was 150 mg/mL,the purification effect was the best under this condition. Also,walnut protein hydrolysates can be divided into the four different components by Sephedax G–15,the highest components of ACE inhibitory activity was 90.35±0.25%,whose IC50 value was 0.158 mg/mL. It found that the peptides effectively purified when comparing the peptides map by RP–HPLC and they were mainly concentrated on the 10,13,17,21 min location. The hydrophobic amino acids were significantly enriched by the size exclusion chromatography,from which the value of the aromatic acids Tyr,Phe were 10.46%and 20.15%respectively.
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