【作者】 蔡丽华；

【导师】 马美湖；

【作者基本信息】 华中农业大学 ， 食品科学， 2010， 硕士

【摘要】 本文利用牛骨蛋白酶解制备血管紧张素转换酶抑制肽,在以下几个方面开展了研究：研究了牛骨软化过程中骨蛋白的变化及实验室制备牛骨粉的营养价值；通过对牛骨的水解发现碱性蛋白酶、中性蛋白酶、胰蛋白酶对牛骨的水解效果较好；研究了酶解条件对牛骨蛋白酶解动力学参数的影响,根据动力学试验得到：碱性蛋白酶作用于骨蛋白的最适温度为55℃、pH为12,此时Vmax为0.10824 g/L·min,Km为11.71892g/L；中性蛋白酶作用于骨蛋白的最适条件温度为45℃、pH为7,此时Vmax为0.04724 g/L·min,Km为14.35711g/L；胰蛋白酶作用于骨蛋白的最适温度为55℃、pH为9,此时Vmax为0.05073g/L·min,Km为4.31516g/L。从新鲜猪肺中提取了血管紧张素转换酶(ACE)的粗酶液,纯化后得到了比活力为5.21u的ACE酶液；通过水解度与ACE抑制率的关系研究发现不同蛋白酶水解的水解度与ACE抑制率的关系不同；在此基础上选用碱性蛋白酶作为酶解骨粉制备ACE抑制肽的工具酶,通过条件的优化得到的最适酶解参数为50℃、pH 10、底物浓度25%、E/S为0.4、酶解时间为3h。利用葡聚糖凝胶色谱测定A3h的分子量分布,发现该混合肽主要分为3个组分,其中组分111分子量小于1000Da,且半抑制浓度最小为0.561mg/ml,抑制活性较高；利用5000Da的中空纤维膜进行超滤,得到的小分子混合肽进行氨基酸分析等定性分析。通过ACE抑制活性消化稳定性实验和ACE抑制动力学研究发现该混合肽为真抑制型的竞争性抑制剂。收集已报道数据进行分析发现肽中疏水性氨基酸和芳香族氨基酸对其ACE抑制活性有较大影响；利用大孔树脂富集疏水性氨基酸,确定了操作条件以50mg/ml骨肽上样浓度为佳,100%乙醇洗脱得到的组分ACE抑制活性最高,其半抑制浓度(IC50)为0.45mg/ml,组分得率为24.59%；利用颗粒活性炭富集芳香族氨基酸,确定了30mg/ml上样浓度为佳,利用100%乙醇+5%乙酸洗脱得到的组分IC50为0.59mg/ml。更多还原

【Abstract】 In this paper, angiotensin-converting enzyme inhibitory peptides were prepared by hydrolysis cattle bone. Therefore, the work was carried out from the following aspects:The changes of bone protein by softening process and nutritional value of cattle bone meal by preparating in the laboratory were studied. The hydrolysis effect of three kinds of enzymes which were alkaline protease, neutral protease and trypsin were better than others. The bone protease solution conditions impacted on the kinetic parameters. According to the dynamic test, the optimum conditions of alkaline protease acted on the bone protein at 55℃and pH 12 and the kinetic parameters was 0.10824 g/L·min for Vmax and 11.71892g/L for Km. The optimum conditions of Neutral protease was at 45℃and pH 7 and the Vmax was 0.04724g/L·min with Km for 14.35711g/L at the same time. The optimum conditions of Trypsin was at 55℃,pH 9 and this time Vmax for 0.05073g/L·min and Km for 4.31516g/L,The crude enzyme solution of angiotensin-converting enzyme (ACE) was extracted from fresh lung. After purification, the specific activity of ACE enzyme solution was improved to 5.21u. The relationship between the degree of hydrolysis and the rate of ACE inhibition was researched. The results were that the regulation was inconsistent in different enzymes. On this basis, the alkaline protease was selected as a tool to preparate ACE inhibitory peptide from cattle bone meal. Optimum hydrolysis conditions were obtained with an enzyme to substrate ratio (E:S) of 0.04 for 3 h at 50℃and pH 10.The molecular weight distribution of hydrolysates was determined by Sephadex G-25 chromatography. There are three peaks in which molecular weight of the peakⅢis less than 1000 Da, the half maximal inhibitory concentration (IC50) was determined as 0.561mg/ml. Ultrafiltrate (5000Da) and hydrolysates were qualitative analyzed by amino acid analyzers. Digestive stability test shows that ACE inhibitory activity is stability because the mixture can not be hydrolysis easily by digestive enzyme.ACE inhibition kinetics study found that the ultra-filtrate was a competitive inhibitor.Scatter and non-linear regression model demonstrates that the proportion of hydrophobic amino acids and aromatic amino acids in protein have great impacted on ACE inhibitory activity of hydrolysis.Based on the above conclusions, macroporous resin and activated carbon were used for enrichment of hydrophobic amino acids and aromatic amino acid to improve ACE inhibitory activity of bone peptide. The optimum sample concentration of macroporous resin purification was 50mg/ml.100% ethanol eluted fraction obtained the highest ACE inhibitory activity and its half-inhibitory concentration (IC50) as 0.45mg/ml, the yield of component as 24.59%.The optimum sample concentration of activated carbon purification was 30mg/ml.100% ethanol and 5% acetic acid elution fraction obtained IC50 as 0.59mg/ml.更多还原