连续化酶膜耦合法制备高活性ACE抑制玉米肽研究

发布时间：2019-06-22 19:09

【摘要】：为了利用植物蛋白资源,提高大宗农产品的副产品——玉米黄粉的附加值,本文以玉米黄粉为原料,采用酶解和膜分离耦合技术对连续制备高活性血管紧张素转化酶(ACE)抑制肽的制备工艺进行了优化,采用纳滤技术进行脱盐；建立了碱性蛋白酶Alcalase连续酶解玉米蛋白动力学模型；考察了玉米ACE抑制肽的活性稳定性；用自发性高血压大鼠(SHR)模型研究了玉米肽(CP)体内降血压效果并对其作用机理进行了初步研究；并采用HPLC/MS/MS法对部分玉米肽的一级结构进行了鉴定。主要结果如下： 1.酶膜反应器法动态制备ACE抑制玉米肽工艺优化 Alcalase连续酶解玉米蛋白的最佳工艺为：温度45℃,循环速度5L/min,酶底比1.5%,料液比3%,在此操作条件下制得玉米肽的ACE抑制率为：89.82%,蛋白质回收率为73.48%,平均膜通量为83.52%；产品的ACE抑制率较问歇式水解工艺提高了20%以上。 2.玉米活性肽的超滤分级及纳滤脱盐超滤处理得到的三个玉米肽级份(Mm1 kDa、Mm3 kDa、Mm5 kDa)中,以Mm 3 kDa的玉米肽级份的ACE抑制活性最高,其半抑制浓度最低(IC5o=0.29),相对于仅经过一次超滤处理的Mm5 kDa级分来说,IC5o值降低了四倍。最佳纳滤脱盐操作条件为：在pH值为9,压力为8 bar,循环次数4的操作条件下,对玉米肽进行脱盐,处理后料液的ACE抑制率为86.77%,脱盐率为89.72%,纳滤处理前后玉米肽ACE抑制活性基本保持稳定。 3.连续酶解动力学模型拟合得到了水解度与底物浓度及水解时间的关系式为：参照此方程,可预测其它底物浓度下的水解度随时间的变化趋势；并进一步建立了Alcalase连续水解玉米蛋白过程动力学模型：得到米氏常数 4.玉米肽的稳定性及其体内降血压活性 ①本实验中得到的三种级分Mm 1 kDa, Mm 3 kDa和Mm 5kDa对温度均具有良好的稳定性,能耐受100℃以内的热处理；各级份在pH=8左右稳定性良好。体外模拟消化试验结果显示,玉米肽具有一定的抵抗胃肠道消化酶消化的能力,其中Mm 3kDa级份玉米肽在模拟消化前后,其活性均高于其它两个级分,经过胃肠消化酶消化后,仍能保持90%以上的ACE抑制活性。 ②在短期给药试验中,对SHR灌胃玉米肽及阳性对照卡托普利(Captopril)1h后,与模型对照组相比,玉米肽高低剂量组(100 mg/kg-bw)、中低剂量组(50mg/kg-bw)和低剂量组(25 mg/kg-bw)血压值均极显著降低(P0.01),最大收缩压降幅分别为26.57 mmHg,19.57 mmHg和17.91 mmHg,高剂量组(100mg/kg-bw)降血压效果虽不及Captopril,但其降血压持续时间比Captopril长,从4h开始,高剂量组玉米肽组的降压效果略高于Captopril组,CP各剂量组可维持降血压效果5h左右。 ③在长期给药试验中,对SHR灌胃不同剂量玉米肽及阳性对照卡托普利(Captopril) 30 d后,CP各剂量组和阳性对照组(2 mg/kg-bw Captopril) SHR的血压值,均随着连续给药时间的延长,血压降幅不断增加。连续灌胃30d后,阳性对照组及CP高低剂量组(100 mg/kg-bw)、中低剂量组(50 mg/kg-bw)、低剂量组(25 mg/kg-bw) SHR其收缩压最大降幅分别下降39.39 mmHg、34.45 mmHg、30.95 mmHg和27.49 mmHg;而对血压正常的Wistar-Kyoto大鼠,长期灌胃高剂量(100 mg/kg-bw)玉米肽后血压值无显著变化。 ④Captopril和CP主要通过抑制肺脏和睾丸组织中ACE酶活,起到降血压作用,肺和睾丸是CP和Captopril作用的主要靶器官,且CP高剂量组(100 mg/kg-bw)的抑制效果略高于Captopril (2 mg/kg-bw)。 5.玉米ACE抑制肽的结构初探经HPLC-MS/MS在线分离,结合蛋白质数据库信息比对,新鉴定了三种源于Mm3kDa级分的玉米肽,其氨基酸序列分别为QQLLPF、QQFLPF和QLLPF,这三种小肽均富含疏水性氨基酸,氮端和碳端的前三个氨基酸均相同,用现有的构效关系理论分析,均为ACE抑制肽。
[Abstract]:in ord to utilize that vegetable protein resource and to improve the additional value of the by-product of the large agricultural product _ corn yellow powder, the preparation technology of the high-activity angiotensin-converting enzyme (ACE) inhibitory peptide is optimized by using the corn yellow powder as a raw material, and the enzymatic hydrolysis and the membrane separation coupling technology are adopted, Desalination is carried out by nanofiltration technology, and the dynamic model of the corn protein of the alkaline protease Alcalase is established, and the activity stability of the ACE inhibitory peptide of the maize is studied. In this paper, the effect of corn peptide (CP) in vivo and its mechanism of action were studied in the model of spontaneously hypertensive rat (SHR), and the primary structure of the partial corn peptide was identified by HPLC/ MS/ MS. The main results are as follows: 1. Enzyme membrane reactor method for dynamically preparing ACE-inhibiting corn peptide technology The optimum process for optimizing the corn protein of the Alcalase continuous enzyme is as follows: the temperature is 45 DEG C, the circulating speed is 5 L/ min, the enzyme bottom ratio is 1.5%, the feed liquid is 3%, and the ACE inhibition of the corn peptide is prepared under the operation condition. The production rate was 89.82%, the recovery of protein was 73.48%, the average membrane flux was 83.52%, and the ACE inhibition rate of the product increased by 20%. % or more.2. Ultrafiltration of the corn active peptide The three corn peptide fractions (Mm1 kDa, Mm3 kDa, Mm5 kDa) obtained by the stage and nanofiltration desalination ultrafiltration are the highest in the ACE inhibitory activity of the Mm 3 kDa corn peptide fraction, and the half inhibitory concentration thereof is the lowest (IC5 o = 0.29), IC5 relative to the Mm5 kDa fraction treated only once The optimum nanofiltration desalting operation conditions are as follows: under the operating conditions with pH value of 9, pressure of 8 bar and cycle number 4, the corn peptide is desalted, the ACE inhibition rate of the treated material liquid is 86.77%, the desalting rate is 89.72%, and the corn peptide ACE inhibition activity before and after the nanofiltration treatment The sex is basically stable. The relationship between the degree of hydrolysis and the concentration of the substrate and the time of hydrolysis is obtained by fitting the kinetic model of the continuous enzyme. The variation trend of the degree of hydrolysis with time can be predicted with reference to this equation, and the continuous hydrolysis of Alcalase is further established. maize protein process Dynamic model: the Michaelis constant of 4. The corn was obtained. the stability of the peptide and the in vivo blood pressure-lowering activity of the peptide are better than the three fractions Mm 1 kDa, the Mm 3 kDa and the Mm 5 kDa, which are obtained in the experiment, and can withstand the heat treatment within 100 DEG C; The results of in vitro simulation and digestion show that the corn peptide has a certain ability to resist the digestive enzyme digestion of the gastrointestinal tract, and the activity of the Mm 3 kDa fraction of the corn peptide before and after the simulated digestion is higher than that of the other. The two fractions, after digestion by the gastrointestinal digestive enzymes, can still The ACE inhibitory activity of more than 90% was maintained. In the short-term administration test, the corn peptide and the positive control captopril (Captopril) were given intragastric administration of the corn peptide and the positive control captopril for 1 hour, and the high and low dosage groups (100 mg/ kg-bw), the middle and low-dose groups (50 mg/ kg-bw) and the low-dose group (25 mg/ kg-bw) were compared with the model control group. The decrease of blood pressure was significantly lower (P 0.01), the maximum systolic pressure was 26.57 mmHg, 19.57 mmHg and 17.91 mmHg, but the blood pressure of high-dose group (100 mg/ kg-bw) was lower than that of Captopril, but the duration of blood pressure reduction was longer than that of Captopril, and the blood pressure-lowering effect of high-dose group of corn peptide was slightly higher than that of Captopril, C. In the long-term administration, the blood pressure values of different doses of the corn peptide and the positive control captopril in the SHR group and the positive control group (2 mg/ kg-bw Captopril) were observed in the long-term administration. The maximum decrease of systolic blood pressure was 39.39 mmHg, 34.45 mmHg, 30.95 mmHg and 27.49 mmHg in low-dose group (100mg/ kg-bw), low-dose group (50 mg/ kg-bw) and low-dose group (25 mg/ kg-bw), while the maximum decrease of systolic blood pressure was 39.39 mmHg, 34.45 mmHg, 30.95 mmHg and 27.49 mmHg, respectively. Normal Wistar-Kyoto rats with normal blood pressure, long-term intragastric high dose (100 mg / kg-bw) There was no significant change in blood pressure after the corn peptide. Captopril and CP were mainly used to inhibit the activity of ACE enzymes in the lung and testis tissues, which played a role in lowering blood pressure. The lung and the testis were the main target organs of CP and Captopril, and the inhibitory effect of the CP high dose group (100 mg/ kg-bw) was slightly higher. 楂樹簬Captopril (2 mg (kg-bw).5. The structure of the ACE inhibitory peptide of maize was isolated by HPLC-MS/ MS, and three corn peptides derived from the Mm3kDa fraction were identified. The amino acid sequence was QQLLPF, QQ, respectively. the three small peptides are rich in the first three amino acids of the hydrophobic amino acid, the nitrogen end and the carbon end,
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：TQ464.7

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