酪蛋白水解物中ACE抑制肽分离及氨基酸序列分析  

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

作　　者：胡志和[1,2] 夏磊[1] 孙振刚[3] 武文起 冯永强[3] 薛璐[1,2] 刘蓄瑾 贾莹[1] 

机构地区：[1]天津商业大学生物技术与食品科学学院,天津300134 [2]天津市食品生物技术重点实验室,天津300134 [3]天津海河乳业有限公司,天津300402

出　　处：《食品科学》2015年第24期156-163,共8页Food Science

基　　金：天津市科技支撑项目(14ZCZDNC00017);天津市高等学校创新团队项目(TD12-5049);天津市应用基础与前沿技术研究计划重点项目(14JCZDJC34500);天津市北辰区科技计划补助项目(2014-CXYH-KF-001)

摘　　要：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、l1.75 μg/mL和421 μg/mL.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. LC- MS/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 Ⅱcontained eight amino acids including valine, serine, proline, leucine, phenylalanine, glutamatic acid, asparaginic acid, and tyrosine; while component Ⅲcontained trace amounts of serine and tyrosine. Eight fragments were obtained from both components by LC-MC/MC analysis, including three ACE inhibitory fragments as2-f （56-57）：YS, as2-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.

关 键 词：牛乳酪蛋白 胃蛋白酶 胰蛋白酶 血管紧张素转化酶 抑制肽 

分 类 号：TS252.42[轻工技术与工程—农产品加工及贮藏工程]