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.     

Activities and sequences of the angiotensin I‐converting enzyme (ACE) inhibitory peptides obtained from the digested lentil (Lens culinaris) globulins

The aim of this study was the identification of potentially bioaccessible ACE‐inhibitory peptides obtained by in vitro gastrointestinal digestion of lentil globulins. ACE‐inhibitory peptides were purified by ion exchange chromatography and gel filtration. After the first step of purification, three peptide fractions with potential antihypertensive properties were obtained and the highest inhibitory activity was determined for the fraction 5 (IC₅₀ = 0.02 mg mL^?1). This fraction was separated on Sephadex G10, and six peptide fractions were obtained. The peptides of fraction (5‐F) with the highest potential antihypertensive activity (IC₅₀ = 0.13 mg mL^?1) were identified using ESI‐MS/MS. The sequences of peptides were KLRT, TLHGMV and VNRLM. Based on Lineweaver–Burk plots for the fraction 5‐F, the kinetic parameters as K_m (1.24 mm ), V_max (0.012 U min^?1), K_i (0.12 mg mL^?1) and mode of inhibition were determined.     

苋籽ACE抑制肽的大孔树脂分离纯化

比较6种大孔树脂对苋籽ACE抑制肽的吸附-解吸效果,从中筛选出合适该活性肽分离纯化的树脂,并对其吸附-解吸工艺进行优化。结果表明,DA201-C树脂最适合苋籽ACE抑制肽的纯化,在样品质量浓度10mg/mL,pH为5,上样量1BV,流速6mL/min时,树脂的吸附效果最佳,吸附率达83.69%,再用5BV体积分数75%乙醇,以5mL/min的流速进行洗脱,此时几乎把吸附的多肽全部洗脱下来,解吸率为98.69%。经树脂纯化,样品的蛋白纯度为89.47%,脱盐率为88.86%,短肽含量提高了20.96%,ACE抑制活性提高了27.91%。      

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。     

The impact of fermentation and in vitro digestion on the formation of angiotensin-I-converting enzyme inhibitory activity from pea and whey protein

Pea and whey protein were fermented by Lactobacillus helveticus and Saccharomyces cerevisiae in monoculture and in combination at 28 and 37 degrees C in order to release angiotensin-I-converting enzyme (ACE) inhibitory peptides. The fermentation products were subjected to in vitro gastrointestinal digestion, and the digests of nonfermented samples served as controls. After fermentation, the ACE inhibitory activity (%) increased by 18 to 30% for all treatments, except for the fermentations of whey protein with Saccharomyces cerevisiae at 28 degrees C, where no significant change was observed. After digestion, however, both fermented and nonfermented samples reached maximum ACE inhibitory activity. The whey digests tended to have lower (50%) inhibitory concentrations (IC50; 0.14 to 0.07 mg/ml), hence, higher ACE inhibitory activity, than the pea digests (0.23 to 0.11 mg/ml). The nonfermented whey protein digest showed the highest ACE inhibitory activity of all. For pea protein, the nonfermented sample had the lowest IC50 value. These results suggest that in vitro gastrointestinal digestion was the predominant factor controlling the formation of ACE inhibitory activity, hence, indicating its importance in the bioavailability of ACE inhibitory peptides.     

益生菌发酵制备金针菇血管紧张素转化酶抑制肽

为了获得高活性的金针菇血管紧张素转化酶（ACE）抑制肽,该研究对益生菌发酵制备金针菇ACE抑制肽的工艺条件进行了优化,并采用超滤法对ACE抑制肽进行了分级分离和活性鉴定。试验以ACE抑制率为评价指标,对枯草芽孢杆菌（Bacillus subtilis）、酿酒酵母（Saccharomyces cerevisiae）和黑曲霉（Aspergillus niger）三种益生菌进行了筛选,优选出沉淀分离活性肽的最适pH,然后采用单因素和响应面设计对发酵条件进行了考察。结果表明,确定枯草芽孢杆菌为最适菌种;沉淀ACE抑制肽的最适pH为6;最佳发酵条件为蒸馏水与金针菇粉液料比5∶1(mL∶g)、发酵时间16 h、发酵温度37℃、金针菇粉用量35 g/500 mL三角瓶,于此条件下ACE抑制率实测值为（51.25±1.02）%;截留分子质量<3 kDa的ACE抑制肽活性最强。     

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。     

Angiotensin converting enzyme (ACE) inhibitory,antihypertensive and antihyperlipidaemic activities of protein hydrolysates from Rhopilema esculentum

Angiotensin-converting enzyme (ACE) inhibitory, antihypertensive and antihyperlipidaemic activities of protein hydrolysates (RPH) from the jellyfish Rhopilema esculentum were investigated. R. esculentum was hydrolysed sequentially with pepsin and papain, and then the hydrolysate was ultrafiltered with a 2000 Da cut-off membrane. It was found that RPH contained high levels of Gly, Glu, Pro, Asp and Ala, having potential ACE inhibitory activity in vitro with an IC₅₀ of 1.28 mg/ml. It was also found that systolic blood pressure was reduced markedly in spontaneously hypertensive rats after single and chronic oral administration of RPH, indicating that RPH had an antihypertensive effect. In addition, oral administration of RPH decreased total serum cholesterol and triglyceride, and increased high-density lipoprotein cholesterol in rats fed with high-fat diet. These results indicate that RPH may prove to be a promising functional food for the prevention and treatment of hypertension and hyperlipidaemia.     

Novel probiotic-fermented milk with angiotensin I-converting enzyme inhibitory peptides produced by Bifidobacterium bifidum MF 20/5

In previous research, we have demonstrated that Bifidobacterium bifidum MF 20/5 fermented milk possessed stronger angiotensin converting enzyme (ACE) inhibitory activity than other lactic acid bacteria, including Lactobacillus helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP). The aim of this study is to investigate the ACE-inhibitory peptides released in B. bifidum MF 20/5 fermented milk. The novel ACE-inhibitory peptide LVYPFP (IC₅₀ = 132 μM) is reported here for the first time. Additionally, other bioactive peptides such as the ACE-inhibitor LPLP (IC₅₀ = 703 μM), and the antioxidant VLPVPQK were identified. Moreover, the peptide and amino acid profiles, the ACE-inhibitory activity (ACEi), pH, and degree of hydrolysis of the fermented milk were determined and compared with those obtained in milk fermented by L. helveticus DSM 13137. The sequences of the major bioactive peptides present in fermented milk of B. bifidum and L. helveticus were identified and quantified. B. bifidum released a larger amount of peptides than L. helveticus but no IPP or VPP were detected in B. bifidum fermented milk. Also the lactotripeptide concentrations and ACEi were higher in L. helveticus fermented milk when the pH was maintained at 4.6. This may represent a technical advantage for B. bifidum that reduces the pH at a slow enough rate to facilitate the peptide generation without the need for pH control. Thus these findings show the potential for the use of this probiotic strain to produce fermented milk with a wider range of health benefits including reduction of blood pressure.     

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。     

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.     

苋籽ACE抑制肽降血压的体外评价

利用循证医学,依据血管紧张素转换酶(ACE)对血压的调节机理,以呋喃丙烯酰三肽(FAPGG)为血管紧张素Ⅰ模拟物,建立了胰蛋白酶模拟体外消化体系和大鼠肝脏混合功能氧化酶(MFO)模拟肝脏代谢体系,测得了未经处理的苋籽水解肽IC50值为1mg/mL,经过体外模拟消化和孵育处理后的苋籽水解肽IC50值为10mg/mL。      

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     

具有ACE抑制活性乳酸菌菌株的筛选与鉴定

以ACE抑制活性和蛋白水解活性为检测指标,选择138株乳酸菌为出发菌株,筛选出具有强ACE抑制活性的乳酸菌菌株.结果表明,筛选出具有强ACE抑制活性的4株乳酸菌,其中3株菌为瑞士乳杆菌,1株菌为干酪乳杆菌.瑞士乳杆菌KLDS1.0485和干酪乳杆菌KLDS1.0486比例为1:1,制成的发酵乳ACE抑制活性可达到61.55%.因此,组合瑞士乳杆菌KLDS1.0485和干酪乳杆菌KLDS1.0486可作为制备乳源ACE抑制肽的优良菌株.     

Antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of cocoa (Theobroma cacao L.) autolysates

The present study investigated antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of cocoa autolysates. After removal of cocoa fat, alkaloids and polyphenols, the remaining proteinous powder was autolyzed at pH 3.5 and 5.2. At similar concentrations, autolysates produced at pH 3.5 indicated the highest reducing power and ACE inhibition activity. However, those generated at pH 5.2 showed the highest antioxidant activity based on ??-carotene bleaching assay. The results displayed a dose-dependent trend. Based on amino acids composition, slight differences were detected between autolysates, and as it was found, they were rich in hydrophobic amino acids. Qualitative and quantitative tests were applied to assure that the results from the assays were not due to the polyphenols of cocoa autolysates. Based on the results no polyphenols could be detected from cocoa autolysates. It can be indicated that among other useful substances of cocoa, its peptides and amino acids could contribute to its health-promoting properties. Furthermore, these bioactive substances can be exploited into functional foods or used as a source of nutraceuticals.     

Influence of adjunct cultures on angiotensin‐converting enzyme (ACE)‐inhibitory activity,organic acid content and peptide profile of kefir

The angiotensin‐converting enzyme (ACE)‐inhibitory activities, peptide profiles and organic acid contents in kefir produced by kefir grains plus lactic acid bacteria as adjunct cultures were determined. All the kefir samples showed almost similar peptide profiles as detected by RP‐HPLC, but quantitative differences were observed during storage. The ACE‐inhibitory activities of different lactic cultures did not exhibit a linear tendency during storage period. After 7 days of storage, there was a significant increase in ACE‐inhibitory activity of the sample fermented with Lactobacillus helveticus. However, a kefir sample containing Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis exhibited a higher ACE‐inhibitory activity (92.23%) compared to the other samples.     

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.