本文關(guān)鍵詞：魔芋ACE抑制肽分離純化及體外降血壓活性研究　出處：《陜西科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 魔芋飛粉 蛋白酶 ACE抑制肽 分離純化 降血壓活性

【摘要】：ACE抑制肽(Angiotensin Converting Enzyme Inhibitory Peptides)是一種對(duì)ACE活性具有抑制作用的多肽類物質(zhì)。ACE抑制肽通過競爭性的與ACE發(fā)生結(jié)合,能夠阻斷具有升血壓活性的血管緊張素II生成,促進(jìn)具有降血壓活性的舒緩肽和腦啡肽生成,從而起到降血壓作用。ACE抑制肽具有分子量小、生物利用度高、對(duì)人體無過敏性等特點(diǎn)。目前,ACE抑制肽的來源和制備方法已經(jīng)成為國內(nèi)外研究的熱點(diǎn)之一。據(jù)報(bào)道,我國魔芋精粉年產(chǎn)量已超過5000噸,在魔芋精粉加工過程中會(huì)產(chǎn)生重量輕、顆粒小的粉末,稱為魔芋飛粉,約為精粉產(chǎn)量的30%~40%。魔芋飛粉大多被直接廢棄掉,只有極少一部分被利用。研究表明,魔芋飛粉中含有豐富的蛋白質(zhì),直接酶解魔芋蛋白質(zhì)可獲得ACE抑制肽。因此,魔芋飛粉有可能是一種制備ACE抑制肽的天然藥物新資源。本文以魔芋飛粉為原料,采用堿溶酸沉法提取魔芋蛋白質(zhì),直接酶解魔芋蛋白質(zhì)制備ACE抑制肽。采用超濾法、Sephadex G-15柱層析及反相高效液相色譜法(RP-HPLC)對(duì)酶解液進(jìn)行分離純化,得到了純度較高的魔芋ACE抑制肽,并且對(duì)其進(jìn)行體外降血壓活性檢測及結(jié)構(gòu)分析。研究內(nèi)容及結(jié)果主要有:(1)采用堿溶酸沉法提取魔芋蛋白質(zhì)。以ACE抑制率和水解度為考察指標(biāo),利用菠蘿蛋白酶、堿性蛋白酶、復(fù)合蛋白酶、風(fēng)味蛋白酶、膠原蛋白酶酶解魔芋蛋白制備ACE抑制肽。通過單因素試驗(yàn)和正交試驗(yàn)優(yōu)化魔芋ACE抑制肽的酶解工藝條件。結(jié)果表明:最佳水解用酶為堿性蛋白酶。在設(shè)定的酶解條件下,酶解p H值對(duì)ACE抑制率影響顯著,酶解溫度、底物濃度、酶用量對(duì)其影響不顯著。得出魔芋ACE抑制肽的最佳酶解工藝條件為:酶解pH為10、底物濃度為3%、酶解溫度為46℃、酶用量5000 U/g、酶解時(shí)間3 h,酶解液的ACE抑制率為82.02%。(2)魔芋ACE抑制肽分離純化。以膜通量和ACE抑制率為考察指標(biāo),采用規(guī)格為10、5、1 KDa的超濾膜進(jìn)行超濾法分離,結(jié)果1 KDa的超濾膜對(duì)魔芋ACE抑制肽的分離效果最好。對(duì)分離條件進(jìn)行優(yōu)化,得到最佳分離條件為:料液pH為9、超濾壓力為0.07 MPa、超濾時(shí)間為15 min,分離后所得粗品的ACE抑制率為85.30%,膜通量為38 L/m2·h。以ACE抑制率為考察指標(biāo),采用Sephadex G-15柱層析對(duì)超濾后的魔芋ACE抑制肽粗品進(jìn)行分離,確定出Sephadex G-15柱層析的最佳分離條件為:樣品濃度為120 mg/mL、流速為0.8 mL/min、上樣量為1.0 mL,分離后組分的ACE抑制率為90.20%。采用反相高效液相色譜法對(duì)Sephadex G-15柱層析分離后的組分進(jìn)行純化,得到峰Ⅰ和峰Ⅱ處兩個(gè)組分,峰Ⅰ處組分的ACE抑制率為24.35%,峰Ⅱ處組分的ACE抑制率為92.85%。采用紫外分光光度法測得純化后樣品中魔芋ACE抑制肽的含量為77.20%。(3)魔芋ACE抑制肽結(jié)構(gòu)分析。運(yùn)用氨基酸分析儀從魔芋ACE抑制肽中檢測出了16種氨基酸,其中精氨酸、谷氨酸、天門冬氨酸含量較高。采用紫外全波長掃描確定魔芋ACE抑制肽的最大吸收波長為218.0 nm,與ACE抑制肽標(biāo)準(zhǔn)品的紫外掃描結(jié)果一致。采用紅外光譜掃描對(duì)魔芋ACE抑制肽樣品進(jìn)行分析檢測,結(jié)果表明:樣品及ACE抑制肽標(biāo)準(zhǔn)品所表現(xiàn)出的紅外光譜特征基本一致,其結(jié)構(gòu)中均存在酰胺鍵、C-C、C-H、O-H、C-N、C=O等基團(tuán)。采用凝膠滲透色譜法對(duì)魔芋ACE抑制肽的分子量及分子量分布進(jìn)行測定,結(jié)果表明其峰值分子量分別為262 Da、634 Da,其分子量分布范圍為:200~700 Da。(4)魔芋ACE抑制肽體外降血壓活性檢測。高效液相色譜法檢測結(jié)果表明:魔芋ACE抑制肽對(duì)ACE的抑制活性為58.90%。紫外分光光度法測得其半數(shù)抑制濃度IC50為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.

【學(xué)位授予單位】：陜西科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R945;R96

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