魔芋ACE抑制肽分离纯化及体外降血压活性研究

发布时间：2018-01-09 01:20

本文关键词：魔芋ACE抑制肽分离纯化及体外降血压活性研究　出处：《陕西科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：ACE抑制肽(Angiotensin Converting Enzyme Inhibitory Peptides)是一种对ACE活性具有抑制作用的多肽类物质。ACE抑制肽通过竞争性的与ACE发生结合,能够阻断具有升血压活性的血管紧张素II生成,促进具有降血压活性的舒缓肽和脑啡肽生成,从而起到降血压作用。ACE抑制肽具有分子量小、生物利用度高、对人体无过敏性等特点。目前,ACE抑制肽的来源和制备方法已经成为国内外研究的热点之一。据报道,我国魔芋精粉年产量已超过5000吨,在魔芋精粉加工过程中会产生重量轻、颗粒小的粉末,称为魔芋飞粉,约为精粉产量的30%~40%。魔芋飞粉大多被直接废弃掉,只有极少一部分被利用。研究表明,魔芋飞粉中含有丰富的蛋白质,直接酶解魔芋蛋白质可获得ACE抑制肽。因此,魔芋飞粉有可能是一种制备ACE抑制肽的天然药物新资源。本文以魔芋飞粉为原料,采用碱溶酸沉法提取魔芋蛋白质,直接酶解魔芋蛋白质制备ACE抑制肽。采用超滤法、Sephadex G-15柱层析及反相高效液相色谱法(RP-HPLC)对酶解液进行分离纯化,得到了纯度较高的魔芋ACE抑制肽,并且对其进行体外降血压活性检测及结构分析。研究内容及结果主要有:(1)采用碱溶酸沉法提取魔芋蛋白质。以ACE抑制率和水解度为考察指标,利用菠萝蛋白酶、碱性蛋白酶、复合蛋白酶、风味蛋白酶、胶原蛋白酶酶解魔芋蛋白制备ACE抑制肽。通过单因素试验和正交试验优化魔芋ACE抑制肽的酶解工艺条件。结果表明:最佳水解用酶为碱性蛋白酶。在设定的酶解条件下,酶解p H值对ACE抑制率影响显著,酶解温度、底物浓度、酶用量对其影响不显著。得出魔芋ACE抑制肽的最佳酶解工艺条件为:酶解pH为10、底物浓度为3%、酶解温度为46℃、酶用量5000 U/g、酶解时间3 h,酶解液的ACE抑制率为82.02%。(2)魔芋ACE抑制肽分离纯化。以膜通量和ACE抑制率为考察指标,采用规格为10、5、1 KDa的超滤膜进行超滤法分离,结果1 KDa的超滤膜对魔芋ACE抑制肽的分离效果最好。对分离条件进行优化,得到最佳分离条件为:料液pH为9、超滤压力为0.07 MPa、超滤时间为15 min,分离后所得粗品的ACE抑制率为85.30%,膜通量为38 L/m2·h。以ACE抑制率为考察指标,采用Sephadex G-15柱层析对超滤后的魔芋ACE抑制肽粗品进行分离,确定出Sephadex G-15柱层析的最佳分离条件为:样品浓度为120 mg/mL、流速为0.8 mL/min、上样量为1.0 mL,分离后组分的ACE抑制率为90.20%。采用反相高效液相色谱法对Sephadex G-15柱层析分离后的组分进行纯化,得到峰Ⅰ和峰Ⅱ处两个组分,峰Ⅰ处组分的ACE抑制率为24.35%,峰Ⅱ处组分的ACE抑制率为92.85%。采用紫外分光光度法测得纯化后样品中魔芋ACE抑制肽的含量为77.20%。(3)魔芋ACE抑制肽结构分析。运用氨基酸分析仪从魔芋ACE抑制肽中检测出了16种氨基酸,其中精氨酸、谷氨酸、天门冬氨酸含量较高。采用紫外全波长扫描确定魔芋ACE抑制肽的最大吸收波长为218.0 nm,与ACE抑制肽标准品的紫外扫描结果一致。采用红外光谱扫描对魔芋ACE抑制肽样品进行分析检测,结果表明:样品及ACE抑制肽标准品所表现出的红外光谱特征基本一致,其结构中均存在酰胺键、C-C、C-H、O-H、C-N、C=O等基团。采用凝胶渗透色谱法对魔芋ACE抑制肽的分子量及分子量分布进行测定,结果表明其峰值分子量分别为262 Da、634 Da,其分子量分布范围为:200~700 Da。(4)魔芋ACE抑制肽体外降血压活性检测。高效液相色谱法检测结果表明:魔芋ACE抑制肽对ACE的抑制活性为58.90%。紫外分光光度法测得其半数抑制浓度IC50为11.09μg/mL。
[Abstract]:ACE (Angiotensin Converting Enzyme Inhibitory inhibitory peptide Peptides) is a kind of ACE can inhibit the activity of polypeptide.ACE inhibitory peptides by competitive binding with ACE, can block the rise of blood pressure with the activity of angiotensin II generation, promote soothing peptides and enkephalin produced antihypertensive activity, thereby the hypotensive effect of.ACE inhibitory peptides have small molecular weight, high bioavailability, no allergic to human. At present, ACE inhibited the sources and the preparation methods of peptide has become one of the hot research at home and abroad. It is reported that China konjac powder production has been more than 5000 tons, in the process of konjac powder will have light weight, small particles of powder, called konjac powder, flour yield is about 30%~40%. konjac powder was mostly discarded directly, only a small part is used. The results show that konjac powder in Rich in protein, direct enzymatic hydrolysis of konjac protein can be ACE inhibitory peptides. Therefore, konjac powder could be a new resource of natural medicine preparation of ACE inhibitory peptides. In this paper, konjac powder as raw material, extraction of konjac protein by alkali solution and acid precipitation, direct enzymatic preparation of ACE inhibitory protein of konjac peptide by ultrafiltration, Sephadex G-15 column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) hydrolysates were isolated and purified by inhibitory peptides with high purity konjac ACE, and analyses its activity and reduced blood pressure in vitro. The research contents and main results are: (1) by alkali extraction of soluble and acid precipitation. The protein konjac ACE inhibition rate and degree of hydrolysis were investigated, using bromelain, alkaline protease, protease, flavor protease, enzyme hydrolysis of collagen konjac protein ACE inhibitory peptides were prepared by single factor test. The process conditions and optimization of ACE inhibitory peptide enzyme konjac orthogonal test. The results showed that the optimum hydrolytic enzyme for alkaline protease hydrolysis conditions. In the setting of enzyme, enzymatic hydrolysis of P H on the ACE inhibition rate significantly, enzymolysis temperature, substrate concentration, enzyme dosage has no significant effect on it. The optimum enzyme inhibition magic taro ACE peptide hydrolysis conditions were: enzyme pH is 10, substrate concentration 3%, hydrolysis temperature 46 C, enzyme dosage 5000 U/g, enzymolysis time 3 h, enzymolysis liquid ACE inhibition rate was 82.02%. (2) of konjac ACE inhibitory peptides were isolated and purified. The membrane flux and the inhibition of ACE rate as an index, the specifications for the membrane ultrafiltration separation of 10,5,1 KDa, the best separation effect of inhibitory peptide 1 KDa ultrafiltration membrane of konjac ACE. The separation conditions were optimized, the optimal separation conditions were: liquid pH was 9, the ultrafiltration pressure is 0.07 MPa, the ultrafiltration time is 15 min after the separation of the crude The ACE inhibition rate was 85.30%, the membrane flux is 38 L/m2 - H. to ACE inhibition rate as indexes of ultrafiltration after konjac ACE inhibitory peptides crude separated by Sephadex G-15 column chromatography, to determine the optimum separation conditions of Sephadex G-15 column chromatography: sample concentration was 120 mg/mL, the flow rate is 0.8 mL/min. The sample volume is 1 mL, after the separation of components of ACE 90.20%. inhibition by reversed-phase high performance liquid chromatography on Sephadex G-15 column chromatography fractions were purified by peak I and peak II at the two components at the peak 1 component of the ACE inhibitory rate was 24.35% at the peak of component ACE 92.85%. inhibition by ultraviolet spectrophotometer was used to measure the content of konjac purification of ACE inhibitory peptide in the sample after 77.20%. (3) peptide structure analysis. Using konjac ACE inhibitory amino acid analyzer from Konjac ACE inhibitory peptide was detected in 16 kinds of amino acid, arginine, glutamine Acid, aspartic acid content is high. The UV wavelength scanning to determine the maximum absorption wavelength of ACE inhibitory peptide konjac is 218 nm, UV scanning results and ACE inhibitory peptide standard. The infrared spectra of konjac ACE inhibitory peptide samples detection, the results show that the infrared spectral characteristics of samples and ACE inhibitory peptide standard showed consistent amide bond existed in the structure of C-C, C-H, O-H, C-N, C=O groups. The molecular weight and molecular weight distribution of konjac ACE inhibitory peptides by gel permeation chromatography were determined, the results showed that the peak molecular weight of 262 Da and 634 Da respectively. And the molecular weight distribution is: 200~700 Da. (4) ACE inhibitory peptides in vitro konjac antihypertensive activity detection. The detection results by HPLC showed that the inhibition of ACE konjac peptide on ACE 58.90%. UV spectrophotometer was used to measure the half inhibition The concentration of IC50 is 11.09 mu g/mL.

【学位授予单位】：陕西科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R945;R96

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