利用鲫鱼加工下脚料酶解制备ACE抑制肽的工艺优化

以鲫鱼加工下脚料为原料,用酶解法制备ACE抑制肽,并以血管紧张素转化酶(angiotension converting enzyme,ACE)抑制率为指标,从碱性蛋白酶、风味蛋白酶、中性蛋白酶、胰蛋白酶、木瓜蛋白酶、胃蛋白酶中筛选出最佳酶解蛋白酶为胃蛋白酶;以单因素试验为基础,应用Box-Benhnken中心组合设计原理和响应面分析法,探讨各自变量及其交互作用对ACE抑制率的影响,通过模拟得到的二次多元式方程预测模型,确定最佳酶解条件为:料液比14.4(mV)、加酶量[E]/[S]=521U/g、酶解时间5.3h。以优化条件制备的酶解产物ACE实际抑制率可达75.79%,与理论预测值76.17%相差不大。     

Purification and characterisation of angiotensin I converting enzyme inhibitory peptides from lysozyme hydrolysates

Hen egg white lysozyme (HEWL) was hydrolysed with trypsin, papain and a combination of the two. The prepared hydrolysates exhibited ACE inhibitory activity. The hydrolysates were fractionated using ultrafiltration and reverse phase-high performance liquid chromatography (RP-HPLC). Three fractions, which showed the highest ACE inhibitory activities, were purified by RP-HPLC. They were the F₇ (from papain-trypsin hydrolysate), F₈ (from papain hydrolysate) and F₃ (from trypsin hydrolysate) fractions. The IC₅₀ values were 0.03, 0.155 and 0.23 mg/ml for F₇, F₈ and F₃, respectively. The F₇ fraction was the most potent ACE inhibitor peptide, and was composed of 12 amino acids, Phe-Glu-Ser-Asn-Phe-Asn-Thr-Gln-Ala-Thr-Asn-Arg (MW: 1428.6 Da). Lineweaver-Burk plots suggest that the F₇ peptide acts as an uncompetitive inhibitor against ACE. The kinetic parameters (K_m, V_max, and K_i) for the F₇ peptide were measured and compared to the control.     

海洋生物来源血管紧张素转换酶抑制肽的研究进展

血管紧张素转化酶(angiotensin converting enzyme,ACE)抑制剂通过抑制ACE活性能够降低血压。目前人工合成ACE抑制药物已开发并应用到降血压的治疗中,但会带来多种副作用。食源性ACE抑制肽与合成抑制剂相比因其副作用小、安全性高等特点,成为ACE抑制肽研究的热点。由于独特的生活环境与巨大的存在数量,海洋生物具有与陆地生物截然不同的化学结构及丰富的生物活性成分。海洋生物来源ACE抑制肽的研究为食源性ACE抑制肽的筛选提供了基础。本文结合海洋生物来源ACE抑制肽的作用机制及制备流程,对鱼类、软体动物、虾类、藻类及海绵、海蜇、粗刺参等不同生物来源的ACE抑制肽制备工艺进行了综述,并对海洋生物的综合开发利用进行了概括。     

食源蛋白制备血管紧张素抑制肽的研究进展

血管紧张素转化酶(angiotensin I converting enzyme,简称ACE)对于人体血压的调节有着十分重要的作用。血管紧张素转化酶抑制肽(ACEIP)是从食源性蛋白质中提取的功能性肽,有着抑制ACE的作用,从而降低人体血压,达到治疗高血压的目的。本文主要从ACE抑制肽的降压原理、来源、结构、分类及作用特点和前景展望这几方面对其进行总结阐述。     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from jellyfish Rhopilema esculentum

Two angiotensin I converting enzyme (ACE) inhibitory peptides were isolated from jellyfish Rhopilema esculentum protein hydrolysates prepared with pepsin and papain. Consecutive purification methods, including ion-exchange chromatography, size-exclusion chromatography and reverse-phase high-performance liquid chromatography, were used for isolation of ACE inhibitory peptides. The amino acid sequence of the peptides was identified as Gln-Pro-Gly-Pro-Thr and Gly-Asp-Ile-Gly-Tyr, respectively, and the IC₅₀ value of the purified peptides for ACE inhibitory activity was 80.67 μM and 32.56 μM. The purified peptides were evaluated for the antihypertensive effect in spontaneously hypertensive rats (SHR) after oral administration. Blood pressure decreased significantly after ingestion of the peptides. The results suggest that peptides derived from jellyfish R. esculentum may be beneficial as antihypertensive compounds in functional food resources.     

血管紧张素转化酶抑制肽稳定性研究

高血压是心血管疾病最重要的且可治疗的危险因素之一,控制高血压是降低心脑血管疾病的发病率、致残率和死亡率的有效措施。血管紧张素转化酶通过影响肾素-血管紧张素系统和激肽释放酶-激肽系统实现对人体血压调节。文章综述了血管紧张素转化酶抑制肽的总体研究趋势、物理化学及酶稳定性的研究现状,并对其值得进一步研究内容进行展望,旨在为血管紧张素转化酶抑制肽的研究与开发提供思路。     

Production of angiotensin I converting enzyme inhibitory (ACE-I) peptides during milk fermentation and their role in reducing hypertension

Fermented milk is a potential source of various biologically active peptides with specific health benefits. Angiotensin converting enzyme inhibitory (ACE-I) peptides are one of the most studied bioactive peptides produced during milk fermentation. The presence of these peptides is reported in various fermented milk products such as, yoghurt, cheese, sour milk, etc., which are also available as commercial products. Many of the ACE-I peptides formed during milk fermentation are resistant to gastrointestinal digestion and inhibit angiotensin converting enzyme (ACE) in the rennin angiotension system (RAS). There are various factors, which affect the formation ACE-I peptides and their ability to reach the target tissue in active form, which includes type of starters (lactic acid bacteria (LAB), yeast, etc.), substrate composition (casein type, whey protein, etc.), composition of ACE-I peptide, pre and post-fermentation treatments, and its stability during gastrointestinal digestion. The antihypertensive effect of fermented milk products has also been proved by various in vitro and in vivo (animal and human trials) experiments. This paper reviews the literature on fermented milk products as a source of ACE-I peptides and various factors affecting the production and activity of ACE-I peptides.     

海洋蛋白源ACE抑制肽研究进展

血管紧张素转化酶(ACE)抑制肽在血压调节中起着重要作用,食源性ACE抑制肽具有较强降血压效果,是调节血压的潜在活性成分。海洋蛋白源ACE抑制肽是食源性活性肽的重要组成部分,是未来研究的热点。本文综述了海洋蛋白源ACE抑制肽的制备、纯化、结构鉴定、体外和体内活性研究及构效关系等研究进展,同时对海洋蛋白源ACE抑制肽的研究和应用前景进行展望。      

Purification and characterisation of angiotensin I converting enzyme (ACE) inhibitory peptides derived from enzymatic hydrolysate of ovotransferrin

Three novel peptides, IQW, IRW and LKP, were predicted in our previous study in the thermolysin–pepsin ovotransferrin hydrolysate. The aims of the present study were to purify the peptides, and determine if the predicted peptides purified from the hydrolysate would have the same activity as the synthetic ones. We also determined the stability of the peptides under simulated gastrointestinal condition. IQW, IRW and LKP were then successfully purified from crude ovotransferrin hydrolysate through multi-step chromatographic purification comprising of cation exchange chromatography followed by three-step reverse-phase high performance liquid chromatography (RP-HPLC), and their sequences were analysed by UPLC-MS/MS. Our results showed that their activities were comparable to the synthetic ones. Simulated gastrointestinal incubation showed that IRW was degraded into a dipeptide of IR and a free amino acid of W by pancreatin, LKP was degraded into a dipeptide of KP and a free amino acid of L by mucosal peptidase, while IQW was stable against the digestive enzymes.     

酪蛋白源ACE抑制肽的精制

采用离子交换树脂对酪蛋白源ACE抑制肽进行脱盐处理,结果表明,以10倍柱体积/h流速,经阴阳离子交换树脂后脱盐率达80%左右,氮回收率为83%,脱盐处理前后基本上对其ACE抑制活性和氨基酸含量没有影响。     

Purification and identification of angiotensin converting enzyme inhibitory peptides from beef hydrolysates

Sarcoplasmic protein extracts from beef rump (biceps femoris) were hydrolyzed (for 0, 4, 8, 12, and 24 h) with three enzymes or their paired combinations. Ultrafiltration, gel-filtration, and RP-HPLC were used to separate angiotensin converting enzyme (ACE) inhibitory peptides from the hydrolysates. The highest ACE inhibitory activity of enzyme hydrolysates resulted from 4 h incubation with enzymes or their paired combinations. The activities of gel filtrated fractions from these hydrolysates were assayed in vitro, demonstrating that the 3rd peak of enzyme thermolysin + proteinase A hydrolysate had the highest ACE inhibition activity (52.8%). The 3rd peak of this hydrolysate was separated by RP-HPLC into five peaks, of which peak 3 showed 30.1% ACE inhibition activity. Its peptide sequence was determined to be Val-Leu-Ala-Gln-Tyr-Lys. The results suggested that this peptide may be a potent ACE inhibitor which might perhaps be used to develop beef with a bioactive peptide to lower blood pressure.     

Purification and hypotensive activity of rapeseed protein-derived renin and angiotensin converting enzyme inhibitory peptides

Rapeseed protein isolate (RPI) was hydrolyzed with Alcalase followed by reverse-phase high performance liquid chromatography (RP-HPLC) purification of bioactive peptides. The rapeseed protein hydrolysate (RPH) obtained after 4 h digestion with Alcalase had a degree of hydrolysis (DH) of ～11%. Gel permeation chromatography separation showed high contents of low molecular weight peptides in the RPH when compared to the RPI. After preparative and analytical RP-HPLC separations, three peptides (LY, TF and RALP) were purified and amino acid sequence determined by tandem mass spectrometry. LY (IC₅₀, 0.11 mM) was the most potent (p < 0.05) against ACE activity when compared to TF (IC₅₀, 0.81 mM) and RALP (IC₅₀, 0.65 mM). However, RALP (IC₅₀, 0.97 mM) was the most potent (p < 0.05) against renin activity when compared to LY (IC₅₀, 1.87 mM) and TF (IC₅₀, 3.1 mM). Single oral administration (30 mg/kg body weight) to spontaneously hypertensive rats showed LY and RALP to be the more effective hypotensive agents with maximum blood pressure reduction of ?26 and 16 mmHg, respectively when compared to TF (?12 mmHg). The results suggest that the higher number of hydrophobic amino acid residues LY and RALP contributed to their higher in vitro and in vivo activities when compared to TF.     

珍珠河蚌肉酶解制备ACE抑制肽研究

采用酸性蛋白酶、中性蛋白酶、碱性蛋白酶、木瓜蛋白酶、胃蛋白酶和胰蛋白酶水解珍珠河蚌肉,通过体外检测方法测定其ACE抑制率。结果表明,胃蛋白酶水解产物的ACE抑制率最大。采用四因素二次通用旋转设计对胃蛋白酶水解河蚌肉的水解条件进行优化,研究了酶与底物的质量比(E∶S)、温度、pH值和时间对水解产物ACE抑制率的影响,建立了回归方程,分析了各因素对ACE抑制率的影响,确定了最优的水解条件。     

The impact of fermentation and in vitro digestion on formation angiotensin converting enzyme (ACE) inhibitory peptides from pea proteins

Pea seeds were fermented by Lactobacillus plantarum 299v in monoculture under different time and temperature conditions and the fermented products were digested in vitro under gastrointestinal conditions. After fermentation and digestion ACE inhibitory activity was determined. In all samples after fermentation no ACE inhibitory activity was noted. Potentially antihypertensive peptides were released during in vitro digestion. The highest DH (68.62%) were noted for control sample, although the lowest IC₅₀ value (0.19 mg/ml) was determined for product after 7 days fermentation at 22 °C. The hydrolysate characterised by the highest ACE inhibitory activity was separated on Sephadex G10 and two peptides fractions were obtained. The highest ACE inhibitory activity (IC₅₀ = 64.04 μg/ml) for the first fraction was noted. This fraction was separated by HPLC and identified by LC–MS/MS and the sequence of peptide derived from pea proteins was determined as KEDDEEEEQGEEE.     

Yeasts from Colombian Kumis as source of peptides with Angiotensin I converting enzyme (ACE) inhibitory activity in milk

This study investigated the possibility of using yeast strains in fermented milks to obtain products with high Angiotensin I-converting enzyme (ACE) inhibitory activity and low bitter taste. Ninety-three yeast strains isolated from Colombian Kumis in different geographic regions were molecularly identified, and their milk fermentation performances were determined. Molecular identification evidenced that Galactomyces geotrichum, Pichia kudriavzevii, Clavispora lusitaniae and Candida tropicalis, were the dominant species. Eighteen out of 93 strains produced fermented milk with ACE-inhibitory (ACEI) activity values ranging from 8.69 to 88.19%. Digestion of fermented milk samples by pepsin and pancreatin demonstrated an increase in ACEI activity, with C. lusitaniae KL4A as the best producer of ACEI peptides. Moreover, sensory analysis of the products containing the major ACE-inhibitory activity pointed out that P. kudriavzevii KL84A and Kluyveromyces marxianus KL26A could be selected as potential adjunct starter cultures in Kumis, since they made a considerable contribution to the ACE inhibitory activity and produced fermented milk without bitter taste. In this study we observed that Colombian Kumis can be an excellent vehicle for the isolation of yeasts with a potential to enhance bioactive peptides produced during milk fermentation.     

Effects of natural peptides from food proteins on angiotensin converting enzyme activity and hypertension

Cardiovascular diseases are the leading cause of death. The underlying pathophysiology is largely contributed by an overactivation of the renin-angiotensin-aldosterone-system (RAAS). Herein, angiotensin II (AngII) is a key mediator not only in blood pressure control and vascular tone regulation, but also involved in inflammation, endothelial dysfunction, atherosclerosis, hypertension and congestive heart failure. Since more than three decades suppression of AngII generation by inhibition of the angiotensin-converting enzyme (ACE) or blockade of the AngII-receptor has shown clinical benefit by reducing hypertension, atherosclerosis and other inflammation-associated cardiovascular diseases. Besides pharmaceutical ACE-inhibitors some natural peptides derived from food proteins reduce in vitro ACE activity. Several animal studies and a few human clinical trials have shown antihypertensive effects of such peptides, which might be attractive as food additives to prevent age-related RAAS activation. However, their inhibitory potency on in vitro ACE activity does not always correlate with an antihypertensive impact. While some peptides with high inhibitory activity on ACE-activity in vitro show no antihypertensive effect in vivo, other peptides with only a moderate ACE inhibitory activity in vitro cause such effects. The explanation for this conflicting phenomenon between inhibitory activity and antihypertensive effect remains unclear to date. This review shall critically address the effects of natural peptides derived from different food proteins on the cardiovascular system and the possible underlying mechanisms. A central aspect will be to point to conceptual gaps in the current understanding of the action of these peptides with respect to in vivo blood pressure lowering effects.     

醋蛋多肽血管紧张素转化酶抑制活性的稳定性研究

研究了温度、pH、金属离子、食盐及食品中常见糖类和防腐剂对醋蛋多肽血管紧张素转化酶(ACE)抑制活性的影响。结果表明:醋蛋多肽的ACE抑制活性对热不稳定,对pH稳定;当金属离子浓度达到5mmol/L时,醋蛋多肽的ACE抑制活性有较明显的下降,其影响大小顺序为K+>Zn2+>Cu2+>Ca2+>Mg2+;食盐能降低醋蛋多肽的ACE抑制活性;在实验浓度范围内,葡萄糖、蔗糖、乳糖、苯甲酸和山梨酸对醋蛋多肽ACE抑制活性影响不大。     

Fractionation of angiotensin I converting enzyme inhibitory activity from pea and whey protein in vitro gastrointestinal digests

In vitro gastrointestinal digestion of pea and whey protein produced high angiotensin I converting enzyme (ACE) inhibitory activity with IC₅₀ values of 0.070 and 0.041 mg protein ml^?1 respectively. Ultrafiltration/centrifugation using a membrane with a molecular weight cut‐off of 3000 Da decreased the IC₅₀ value to 0.055 mg protein ml^?1 for pea permeate and 0.014 mg protein ml^?1 for whey permeate. Further fractionation by reverse phase HPLC gave IC₅₀ values as low as 0.016 mg protein ml^?1 for pea and 0.003 mg protein ml^?1 for whey. Consequently, these purification steps enriched the ACE inhibitory activity of the pea digest more than four times and that of the whey digest more than 13 times. HPLC profiles after digestion and ultrafiltration indicate that high ACE inhibitory activity is due to short and more hydrophobic peptides. The results also suggest that potent ACE inhibitory peptides were present alongside low active peptides in whey hydrolysate, while all peptides had more or less the same ACE inhibitory activity in pea hydrolysate. In addition, the hydrolysates and enriched fractions will resist in vivo gastrointestinal digestion after oral administration. Hence these ACE inhibitory peptides, as part of functional foods, can play significant roles in the prevention and treatment of hypertension. Copyright © 2004 Society of Chemical Industry     

Isolation of angiotensin I converting enzyme (ACE) inhibitor from fermented oyster sauce, Crassostrea gigas

Angiotensin I converting enzyme (ACE) inhibitor was isolated from fermented oyster sauce (FOS) and purified. Oyster was fermented with 25% NaCl (w/w) at 20 °C for 6 months. FOS was passed through a 40-mesh sieve, desalted using an electrodialyzer and then lyophilized. ACE inhibitory activity of FOS was investigated, and the IC₅₀ value was determined to be 2.45 mg/ml. ACE inhibitor from FOS was purified using SP-Sephadex C-25 ion exchange chromatography, Sephadex G-50 gel chromatography, high-performance liquid chromatography (HPLC) on a gel permeation chromatography (GPC) column and reversed-phase HPLC on a C₁₈ column. The purified inhibitor had an IC₅₀ value of 0.0874 mg/ml, and it exhibited competitive inhibition against ACE. The purified peptide was evaluated for its antihypertensive effect in spontaneously hypertensive rats (SHRs) following oral administration. Rat blood pressure significantly decreased after inhibitor injection.