連續(xù)化酶膜耦合法制備高活性ACE抑制玉米肽研究
發(fā)布時(shí)間：2019-06-22 19:09
【摘要】：為了利用植物蛋白資源,提高大宗農(nóng)產(chǎn)品的副產(chǎn)品——玉米黃粉的附加值,本文以玉米黃粉為原料,采用酶解和膜分離耦合技術(shù)對(duì)連續(xù)制備高活性血管緊張素轉(zhuǎn)化酶(ACE)抑制肽的制備工藝進(jìn)行了優(yōu)化,采用納濾技術(shù)進(jìn)行脫鹽；建立了堿性蛋白酶Alcalase連續(xù)酶解玉米蛋白動(dòng)力學(xué)模型；考察了玉米ACE抑制肽的活性穩(wěn)定性；用自發(fā)性高血壓大鼠(SHR)模型研究了玉米肽(CP)體內(nèi)降血壓效果并對(duì)其作用機(jī)理進(jìn)行了初步研究；并采用HPLC/MS/MS法對(duì)部分玉米肽的一級(jí)結(jié)構(gòu)進(jìn)行了鑒定。主要結(jié)果如下： 1.酶膜反應(yīng)器法動(dòng)態(tài)制備ACE抑制玉米肽工藝優(yōu)化 Alcalase連續(xù)酶解玉米蛋白的最佳工藝為：溫度45℃,循環(huán)速度5L/min,酶底比1.5%,料液比3%,在此操作條件下制得玉米肽的ACE抑制率為：89.82%,蛋白質(zhì)回收率為73.48%,平均膜通量為83.52%；產(chǎn)品的ACE抑制率較問(wèn)歇式水解工藝提高了20%以上。 2.玉米活性肽的超濾分級(jí)及納濾脫鹽 超濾處理得到的三個(gè)玉米肽級(jí)份(Mm1 kDa、Mm3 kDa、Mm5 kDa)中,以Mm 3 kDa的玉米肽級(jí)份的ACE抑制活性最高,其半抑制濃度最低(IC5o=0.29),相對(duì)于僅經(jīng)過(guò)一次超濾處理的Mm5 kDa級(jí)分來(lái)說(shuō),IC5o值降低了四倍。最佳納濾脫鹽操作條件為：在pH值為9,壓力為8 bar,循環(huán)次數(shù)4的操作條件下,對(duì)玉米肽進(jìn)行脫鹽,處理后料液的ACE抑制率為86.77%,脫鹽率為89.72%,納濾處理前后玉米肽ACE抑制活性基本保持穩(wěn)定。 3.連續(xù)酶解動(dòng)力學(xué)模型 擬合得到了水解度與底物濃度及水解時(shí)間的關(guān)系式為：參照此方程,可預(yù)測(cè)其它底物濃度下的水解度隨時(shí)間的變化趨勢(shì)；并進(jìn)一步建立了Alcalase連續(xù)水解玉米蛋白過(guò)程動(dòng)力學(xué)模型：得到米氏常數(shù) 4.玉米肽的穩(wěn)定性及其體內(nèi)降血壓活性 ①本實(shí)驗(yàn)中得到的三種級(jí)分Mm 1 kDa, Mm 3 kDa和Mm 5kDa對(duì)溫度均具有良好的穩(wěn)定性,能耐受100℃以?xún)?nèi)的熱處理；各級(jí)份在pH=8左右穩(wěn)定性良好。體外模擬消化試驗(yàn)結(jié)果顯示,玉米肽具有一定的抵抗胃腸道消化酶消化的能力,其中Mm 3kDa級(jí)份玉米肽在模擬消化前后,其活性均高于其它兩個(gè)級(jí)分,經(jīng)過(guò)胃腸消化酶消化后,仍能保持90%以上的ACE抑制活性。 ②在短期給藥試驗(yàn)中,對(duì)SHR灌胃玉米肽及陽(yáng)性對(duì)照卡托普利(Captopril)1h后,與模型對(duì)照組相比,玉米肽高低劑量組(100 mg/kg-bw)、中低劑量組(50mg/kg-bw)和低劑量組(25 mg/kg-bw)血壓值均極顯著降低(P0.01),最大收縮壓降幅分別為26.57 mmHg,19.57 mmHg和17.91 mmHg,高劑量組(100mg/kg-bw)降血壓效果雖不及Captopril,但其降血壓持續(xù)時(shí)間比Captopril長(zhǎng),從4h開(kāi)始,高劑量組玉米肽組的降壓效果略高于Captopril組,CP各劑量組可維持降血壓效果5h左右。 ③在長(zhǎng)期給藥試驗(yàn)中,對(duì)SHR灌胃不同劑量玉米肽及陽(yáng)性對(duì)照卡托普利(Captopril) 30 d后,CP各劑量組和陽(yáng)性對(duì)照組(2 mg/kg-bw Captopril) SHR的血壓值,均隨著連續(xù)給藥時(shí)間的延長(zhǎng),血壓降幅不斷增加。連續(xù)灌胃30d后,陽(yáng)性對(duì)照組及CP高低劑量組(100 mg/kg-bw)、中低劑量組(50 mg/kg-bw)、低劑量組(25 mg/kg-bw) SHR其收縮壓最大降幅分別下降39.39 mmHg、34.45 mmHg、30.95 mmHg和27.49 mmHg;而對(duì)血壓正常的Wistar-Kyoto大鼠,長(zhǎng)期灌胃高劑量(100 mg/kg-bw)玉米肽后血壓值無(wú)顯著變化。 ④Captopril和CP主要通過(guò)抑制肺臟和睪丸組織中ACE酶活,起到降血壓作用,肺和睪丸是CP和Captopril作用的主要靶器官,且CP高劑量組(100 mg/kg-bw)的抑制效果略高于Captopril (2 mg/kg-bw)。 5.玉米ACE抑制肽的結(jié)構(gòu)初探 經(jīng)HPLC-MS/MS在線(xiàn)分離,結(jié)合蛋白質(zhì)數(shù)據(jù)庫(kù)信息比對(duì),新鑒定了三種源于Mm3kDa級(jí)分的玉米肽,其氨基酸序列分別為QQLLPF、QQFLPF和QLLPF,這三種小肽均富含疏水性氨基酸,氮端和碳端的前三個(gè)氨基酸均相同,用現(xiàn)有的構(gòu)效關(guān)系理論分析,均為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(shù)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,
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：TQ464.7
本文編號(hào)：2504883
[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(shù)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,
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：TQ464.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 趙謀明;任嬌艷;;大宗低值蛋白資源精深加工及高值化利用[J];北京工商大學(xué)學(xué)報(bào)(自然科學(xué)版);2010年05期
2 管萍;胡小玲;范曉東;張新麗;岳紅;;納濾膜分離技術(shù)分離純化多肽和氨基酸[J];化學(xué)通報(bào);2006年02期
3 朱宏吉,胡金榜,金鼎五,聶清德,邢義X;動(dòng)態(tài)膜分離式酶反應(yīng)器連續(xù)操作特性研究[J];化工科技;2002年02期
4 劉亞麗,李會(huì)俠,王紅新;酶解玉米渣生產(chǎn)玉米蛋白肽的研究[J];糧食科技與經(jīng)濟(jì);2005年02期
5 周金盛,陳觀文;納濾膜技術(shù)的研究進(jìn)展[J];膜科學(xué)與技術(shù);1999年04期
6 王海濱;生物活性肽的應(yīng)用研究概況[J];肉類(lèi)工業(yè);1999年08期
7 孫鳳祥,曲梅花,高佩林,任維棟;豬肺血管緊張素轉(zhuǎn)換酶的制備[J];中國(guó)生化藥物雜志;2002年03期
8 劉冬;李世敏;張麗君;;玉米降血壓肽對(duì)體內(nèi)外血管緊張素轉(zhuǎn)化酶活性的抑制作用[J];中國(guó)生化藥物雜志;2007年03期
9 陳山,楊曉泉,郭祀遠(yuǎn),蔡妙顏;大豆肽超濾分離純化過(guò)程的研究[J];食品與發(fā)酵工業(yè);2003年01期
10 張永忠,孫艷梅;納濾膜及其在食品工業(yè)中應(yīng)用[J];食品工業(yè);2002年02期
相關(guān)博士學(xué)位論文 前2條
1 黎觀紅;食物蛋白源血管緊張素轉(zhuǎn)化酶抑制肽的研究[D];江南大學(xué);2005年
2 董春華;酪蛋白—胰酶水解動(dòng)力學(xué)模型及反沖與膜表面改性提高酶膜反應(yīng)器性能的研究[D];天津大學(xué);2004年
相關(guān)碩士學(xué)位論文 前2條
1 隋玉杰;玉米肽的制備條件及其醒酒護(hù)肝機(jī)理研究[D];華中農(nóng)業(yè)大學(xué);2006年
2 張貴川;食源三肽ACE抑制活性構(gòu)效關(guān)系研究[D];西南大學(xué);2009年
，本文編號(hào)：2504883
本文鏈接：http://sikaile.net/shekelunwen/minzhuminquanlunwen/2504883.html
最近更新
教材專(zhuān)著
