酪蛋白水解物中ACE抑制肽分离及氨基酸序列分析

以牛乳酪蛋白为底物，先经胃蛋白酶水解，再经胰蛋白酶水解，从水解物中分离获得血管紧张素转化酶（angiotensin converting enzyme，ACE）抑制肽并确定其氨基酸序列。酪蛋白的双酶水解物经超滤和葡聚糖凝胶色谱分离，分离得到3 个组分，收集高ACE活性抑制效果组分Ⅱ和Ⅲ。采用离子色谱法分析水解片段的氨基酸组成，液相色谱-质谱法分析水解片段的氨基酸序列，采用固相合成法制备获得的短肽片段，用分光光度法检测ACE活性抑制效果。结果表明，组分Ⅱ包含缬氨酸、丝氨酸、脯氨酸、亮氨酸、苯丙氨酸、谷氨酸、天冬氨酸、酪氨酸，共8 种氨基酸，组分Ⅲ包含痕量的丝氨酸和酪氨酸；将组分Ⅱ和Ⅲ经液相色谱-质谱分析，获得8 个片段，其中，αs2-f（56～57）∶YS、αs2-f（98～107）∶YQKFPQYLQY和κ-f（52～61）∶INNQFLPYPY具有ACE活性抑制作用，其IC50值分别为11.89、11.75 μg/mL和421 μg/mL。

Separation of ACE Inhibitory Peptides from Casein Hydrolysate and Analysis of Their Amino Acid Sequences

The objectives of this study were to separate angiotensin converting enzyme (ACE) inhibitory peptides from
casein hydrolysate produced by sequential hydrolysis of casein with pepsin followed by trypsin and to analyze their
amino acid sequences. The hydrolysate was concentrated by ultrafiltration and separated by Sephadex G-15 column
chromatography into three components. Components Ⅱ and Ⅲ, which had higher inhibitory effect on ACE activity, were
collected. Ion chromatography was used to analyze amino acid composition of fragments from the two components. LCMS/
MS was used to analyze amino acid sequence of fragments, solid-phase synthesis was used to synthetize short-chain
peptides, and a spectrophotometric method was used to test the inhibitory effect of hydrolysate on ACE activity. Results
showed that component Ⅱ contained eight amino acids including valine, serine, proline, leucine, phenylalanine, glutamatic
acid, asparaginic acid, and tyrosine; while component Ⅲ contained trace amounts of serine and tyrosine. Eight fragments
were obtained from both components by LC-MC/MC analysis, including three ACE inhibitory fragments αs2-f (56-57):YS,
αs2-f (98-107):YQKFPQYLQY and κ-f (52-61):INNQFLPYPY with half maximal inhibitory concentration (IC50) of 11.89,
11.75 and 421 μg/mL, respectively.