红非鲫鱼皮胶原蛋白酶解产物中血管紧张素转换酶抑制肽的分离及其部分特性

背景:从红非鲫鱼皮胶原蛋白酶解产物中分离血管紧张素抑制肽有待研究。目的:通过酶解红非鲫鱼皮胶原蛋白获取具有高血管紧张素转换酶抑制活性的肽。设计:采用正交实验法进行复合酶解,采用超滤、凝胶柱层析和高压液相色谱技术进行降压肽的分离。单位:中国海洋大学水产品高值化利用实验室。材料:体质量(500±50)g红非鲫,由即墨热电厂罗非鱼分厂提供。方法:实验于2003-05/2004-05在中国海洋大学水产品高值化利用实验室完成。①采用稍作改进的Grossman和Bergman法提取红非鲫鱼皮胶原蛋白,通过菠萝蛋白酶和alcalase 2.4L组成的复合酶依次对胶原蛋白进行水解。将红非鲫鱼皮胶原蛋白水解物煮沸使酶失活。得到的红非鲫鱼皮胶原蛋白水解物通过截流相对分子质量分别为6000,4000,1000的超滤膜进行分离,得到3个组分,红非鲫鱼皮胶原蛋白水解物Ⅰ(相对分子质量6000～4000)、红非鲫鱼皮胶原蛋白水解物Ⅱ(相对分子质量4000～1000)和红非鲫鱼皮胶原蛋白水解物Ⅲ(相对分子质量小于1000)。②按照cushman等人的方法测定各组分的血管紧张素转换酶抑制活性。将抑制50%的血管紧张素转换酶活性所需抑制剂浓度定义为IC50。③将具有最高血管紧张素转换酶抑制活性的红非鲫鱼皮胶原蛋白水解物上预先采用1.0 mol/L乙酸缓冲液(内含0.1%v/v吡啶,pH=4.0)平衡的sephadexG-25柱(1.6×100cm),检测波长220nm,对水解原液,RTCH-Ⅰ、RTCH-Ⅱ和RTCH-Ⅲ4个样品进行分离,收集活性峰,对每个活性峰测定其血管紧张素转换酶抑制率。冻干后进一步用反相HPLC ODS-C18分析柱分离。得到各组的高活性组分,同样方法测定其血管紧张素转换酶抑制率。④对4种高活性组分组分的样品均在110℃下用内含1g/L硫代乙醇酸的6 mol/L HCl真空水解,采用氨基酸自动分析仪分析氨基酸组成,采用HPLC法测定各组分的分子量。主要观察指标:①红非鲫鱼皮胶原蛋白水解物的血管紧张素转换酶抑制活性(IC50值)。②血管紧张素转换酶抑制肽的纯化结果。③血管紧张素转换酶抑制肽的氨基酸分析。结果:①红非鲫鱼皮胶原蛋白水解物的血管紧张素转换酶抑制活性:RTCH-Ⅰ,Ⅱ,Ⅲ组分的IC50值分别为2.82,3.30,2.23和0.31 g/L,其中RTCH-Ⅲ的血管紧张素转换酶抑制活性最大。②血管紧张素转换酶抑制肽的纯化结果:4个活性峰的IC50分别为3.5,2.12,1.56和0.65 g/L。采用反相HPLC分析柱(ODS-C18)对6k、4k、1k和ACF组分进一步分离,结果分别如图2、3、4和5所示。实验结果显示,高活性组分分别为Mr6000组分第4个峰以及Mr4000,1000和水解原液的组分第2个峰,其血管紧张素转换酶抑制率分别为11.1%,89.9%,65%和49.7%,其中Mr4000组分第2个峰,显示出最高的抑制活性。③血管紧张素转换酶抑肽的氨基酸分析结果:具有血管紧张素转换酶抑制活性的分离肽产物富含疏水氨基酸和芳香族氨基酸。结论:红非鲫鱼皮胶原蛋白的复合酶水解产物中存在较高活性的血管紧张素抑制肽,该肽富含疏水氨基酸和芳香族氨基酸,可以通过超滤、柱层析、液相色谱法从水解产物中分离。

Separation and partial characterization of agiotensin Ⅰ-converting enzyme inhibitory peptides from enzymic hydrolysates of red tilapia skin collagen

BACKGROUND: Agiotensin Ⅰ -converting enzyme (ACE) inhibitory peptides which are separated from red tilapia skin collagen should be researched further.OBJECTIVE: To obtain a peptide of high ACE inhibitory activity through enzymic hydrolysates of red tilapia skin collagen.DESIGN: Enzymic hydrolysates were done with orthogonal experimental method; decompression peptide was separated with ultrafiltration, gel chromatography and hypertensive liquid chromatography.SETTING: Laboratory of Aquatic Products, Chinese Marine University.MATERIALS: The red tilapia weighing (500+50)g was donated by Branch Factory of Jimo Redian Factory. ACE was isolated from hog lung.METHODS: The experiment was carried out in the Laboratory of Aquatic Products, Chinese Marine University from May Bergman.①The collagen from red tilapia skin was extracted using the method described by Grossman and Bergman.The collagen extraction was hydrolyzed with compound enzymes in the order of bromelain and Alcalase 2.4 L.The red tilapia collagen hydrolysates (RTCH) were subsequently boiled to inactivate the enzyme. The resultant RTCH was fractionated through three different UF membrane bioreactor system having a range of molecular weight cut-offs (MWCO) of Mr 6 000, 4 000 and 1 000. Three portions were obtained: RTCH-I (M, 6000-4000), RTCH- Ⅱ (Mr 4000-1000)and RTCH-Ⅲ(Mr＜1000).②The ACE inhibitory 50%of ACE activity was defined as the IC50 value.③RTCH-Ⅲ(1.5 Ml) with the highest activity among RTCHs was loaded onto a Sephadex G-25 column (1.6×100 cm), and the absorbance of theeluent was monitored at 220 nm. Four samples of primary hydrolysates, RTCH- Ⅰ, RTCH- Ⅱ and RTCHⅢ were separated to collect active fractions which were pooled and lyophilized, immediately. The lyophilized fraction was separated with HPLC ODS-C18 analysis column to obtain component of high activity. Meanwhile, the same method was used to measure ACE inhibitory rate.④Each sample was hydrolyzed with 6 mol/L hydrochloric acid containing 1g/L thioglycolic acid at 110 ℃ for 24 hours in vacuum. The amino acid compositions of the resultant hydrolysates were determined on an amino acid auto analyzer, and molecular weight was measured with HPLC technique.MAIN OUTCOME MEASURES：①ACE inhibitory activity of fractionated RTCHS;②Purification of ACE inhibitory peptide;③Amino acid analysis of ACE inhibitory peptides.RESULTS:①ACE inhibitory activityof fractionated RTCHs:IC50values of RTCH-Ⅰ,RTCH-Ⅱand RTCH-Ⅲ were 3.30,2.23 and 0.31 g/L,and inhibitory of RTCH-Ⅲ was the highest.②Purification of ACE inhibitory peptide: The IC50 value of the four peak were 3.5, 2.12, 1.56 and 0.65 g/L, respectively. Results in Figures 2, 3, 4 and 5 showed that the high active fractions were: 6K4, 4K2, 1K2 and ACF2, and the inhibitory ratio were 11.1%, 89.9%, 65.0% and 49.7%, respectively.Among of these fractions,the 4K2 exhibited the highest inhibitory rate.③Amino acid analysis of ACE inhibitory peptides: Separated peptide products had more aromatic and hydrophobic amino acids.CONCLUSTON: Enzymic hydrolysates of red tilapia skin collagen have high ACE inhibitory peptide which is separated with ultrafiltration, gel chromatography and hypertensive liquid chromatography.