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     

产ACE抑制肽乳酸菌的筛选、益生特性评定及应用

目的:筛选出富产血管紧张素转化酶(Angiotensin converting enzyme,ACE)抑制肽的乳酸菌并评定其益生特性.方法:依据蛋白水解度及ACE抑制率对乳酸菌进行初筛,然后依据模拟胃肠消化后的ACE抑制率最终筛出2株乳酸菌ZJUIDS09和ZJUIDS11,进一步评价其耐酸、耐胆盐、抗生素耐药性和抑菌...     

Profiles,antioxidative and ACE inhibitory activity of peptides released from fermented buttermilk before and after simulated gastrointestinal digestion

Bioactive peptides, released from buttermilk by fermentation and/or gastrointestinal proteases, may have health promoting effects. Thus, a comprehensive analysis of the peptide fraction of fermented buttermilk, before and after different phases of simulated gastrointestinal digestion, was performed using ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS). Results showed that digestion simulation substantially changed the peptide profile of fermented buttermilk. A total of 81, 120 and 46 peptides were identified in fermented buttermilk, its gastric and intestinal digests, respectively. These peptides released mostly from β-casein followed by αs1-casein, κ-casein and β-lactoglobulin. In addition, 14 peptides released from milk fat globule membrane proteins (lactadherin, butyrophilin and GlyCAM-1). Bioactivity, mainly angiotensin converting enzyme (ACE) inhibitory activity, has been reported before for only 54 of the detected peptides. Radical scavenging, ferric reducing and ACE inhibitory activities of fermented buttermilk peptides increased significantly after digestion, indicating promotion in fermented buttermilk-peptide bioactivity by gastrointestinal digestion.     

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.     

动植物源蛋白体外消化产物结构性质及ACE抑制活性

为揭示动植物源蛋白对于血压调节功能上的差异是否由蛋白肽的血管紧张素转化酶（angiotensin conversion enzyme,ACE）抑制活性所引起,动植物蛋白经体外消化后,对蛋白肽的ACE活性抑制进行研究。采用胃蛋白酶-胰蛋白酶两步消化法,水解植物蛋白（大米、燕麦、大豆、豌豆）、红肉蛋白（猪肉、牛肉）和白肉蛋白（鸡肉）。测定所获蛋白肽的水解度、分子质量分布、氨基酸组成及其ACE抑制率。结果表明,随着蛋白质水解度的增加和分子质量的减小,3类蛋白消化产物的ACE活性抑制率均相应增大。其中,植物蛋白消化产物ACE抑制率最高（60.41%）,红肉蛋白最低（40.13%）。对蛋白质消化产物的氨基酸组成进行分析,结果表明,植物蛋白和白肉蛋白消化产物的疏水性氨基酸含量均显著高于红肉蛋白。3类蛋白中消化产物的疏水性氨基酸含量越高,则ACE抑制活性越高,说明疏水性氨基酸含量高可能是植物蛋白肽具有高的ACE抑制活性的主要原因之一。     

发酵酸化技术对乳饼蛋白质降解及蛋白肽生物活性的影响

通过发酵酸化技术研制发酵型乳饼,以传统的直接酸化技术为对照,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）、液相色谱-质谱测定乳饼蛋白质降解情况和游离氨基酸含量,通过用液相色谱-串联质谱鉴定乳饼蛋白肽。并通过测定体外抗氧化活性、α-葡萄糖苷酶抑制率和血管紧张素转换酶（angiotensin-converting enzyme,ACE）抑制率表征乳饼蛋白肽的生物活性。SDS-PAGE结果表明,两种酸化技术均能促进乳饼蛋白质的降解,发酵酸化技术对乳清蛋白的降解程度更高,能促进游离氨基酸的释放。从发酵酸化乳饼中鉴定出潜在的ACE抑制肽23?条、抗氧化肽6?条和降糖肽5?条,而传统的直接酸化乳饼中分别为13、4?条和1?条。发酵酸化技术能显著提升乳饼蛋白肽的α-葡萄糖苷酶抑制活性和ACE抑制活性（P＜0.05）,其中分子质量＜3?kDa超滤组分对二者抑制率的IC50分别为1.250?mg/mL和0.416?mg/mL。发酵酸化技术能促进乳饼蛋白质降解,释放生物活性多肽,同时产生游离氨基酸,提升乳饼的生物学价值。     

瑞士乳杆菌发酵乳清蛋白制备ACE抑制肽的条件优化

对瑞士乳杆菌发酵乳清蛋白产生血管紧张素转化酶(angiotensin converting enzyme,ACE)抑制肽的发酵条件进行探讨。通过单因素分析和响应面试验设计,确定发酵乳清蛋白制备ACE抑制肽的最佳工艺条件：发酵时间为17.52h,发酵温度为37.07℃,乳清质量浓度为114.1g/L,其ACE抑制率可达89.337%。     

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.     

酶解与体外模拟胃肠消化对脱脂羊奶粉ACE抑制活性的影响

本研究旨在探究商业蛋白酶酶解与体外模拟胃肠消化对高消化率的脱脂羊奶粉释放ACE抑制肽的影响,以评价脱脂羊奶粉酶解必要性及所需酶解程度。采用中性蛋白酶、碱性蛋白酶、复合蛋白酶和风味蛋白酶对脱脂后的圭山羊奶粉进行酶解反应,并对脱脂羊奶粉及其酶解物进行体外模拟胃肠消化,测定酶解液及模拟胃肠消化液的多肽含量、ACE抑制活性以及分子量分布。研究发现,羊奶粉经过体外模拟胃肠消化后,消化液的多肽含量与ACE抑制率分别达到20.81 mg/mL和62.55%;而经过碱性蛋白酶和风味蛋白酶的适度酶解,其模拟胃肠消化液多肽含量与ACE抑制活性反而下降;经过复合蛋白酶的适度酶解,其模拟胃肠消化液的多肽含量与ACE抑制活性小幅度提高,分别达到22.67 mg/mL和69.29%;经过中性蛋白酶的适度酶解,其模拟胃肠消化液多肽含量与ACE抑制活性均显著提高,分别达到23.76 mg/mL和81.10%。分子量分布结果显示,经过体外模拟胃肠消化后,<1000 Da的小分子肽由酶解液中的70.77%提高到90%。综上,圭山羊奶粉经过体内胃肠消化就可以释放出较多的ACE抑制肽,而经过中性蛋白酶酶解至水解度8%后...     

源于鸡蛋清溶菌酶ACE抑制肽的分离纯化及结构鉴定

利用胃肠消化酶系水解疏水性氨基酸含量高的蛋清溶菌酶蛋白,筛选能抗胃肠消化的高活性ACE抑制肽。通过超滤、制备型反相高效液相色谱(RP-HPLC)、分析型RP-HPLC纯化、氨基酸组成分析及基质辅助激光解吸电离飞行时间串联质谱(MALDI-TOF-TOF-MS),从胃肠消化水解液中分离鉴定出两种ACE抑制肽Lys-Val-Phe(KVF)和Trp-Ile-Arg(WIR),化学合成该两种三肽后,ACE抑制活性测定结果显示其IC50值分别为14μmol/L与88.5μmol/L。研究结果表明,蛋清溶菌酶蛋白来源的ACE抑制肽有望用于高血压的预防与治疗。     

Preparation of angiotensin-I-converting enzyme inhibitory hydrolysates from unsupplemented caprine whey fermentation by various cheese microflora

Unsupplemented caprine whey was fermented by 25 cheese microflora in order to produce peptides from α-lactalbumin (α-la) and/or β-lactoglobulin (β-lg) hydrolysis. Fourteen hydrolysates enriched in peptides mainly released from α-la were obtained. Angiotensin-I-converting enzyme (ACE) inhibitory activity of each hydrolysate was investigated. Six of them had high ACE inhibitory activities ranging from 31% to 56%. The highest ACE inhibitory activity was obtained after whey fermentation by the microflora from 18 months ripened Comté cheese. The microflora was identified as a co-culture of Candida parapsilosis and Lactobacillus paracasei. Hydrolysate activity remained stable after pepsin, trypsin and chymotrypsin treatments simulating an in vitro gastrointestinal digestion. This hydrolysate was further fractionated by RP-HPLC. The peptide exhibiting the highest ACE inhibitory activity was characterised as WLAHK (α-la f(104–108): Trp–Leu–Ala–His–Lys). WLAHK was resistant toward pepsin and trypsin treatments but was digested by chymotrypsin.     

发酵乳中血管紧张素转换酶抑制剂的分离与鉴定

用瑞士乳杆菌生产具有抑制血管紧张素转换酶(ACE)活性的发酵乳并从其中分离血管紧张素转换酶抑制剂(ACEI)。脱脂乳经灭菌后,接种4%(V/V)的发酵剂,37℃培养24h,获得抑制ACE活性较高的发酵乳。对此发酵乳的乳清进行超滤、上海732阳离子树脂交换、SephadexG-15凝胶过滤和HPLC分步纯化,最终得到两种起主要作用的ACEI肽。经氨基酸分析和液相-质谱分析,这两种肽的氨基酸序列分别为:Phe-Pro(IC50:12.05μmol/L)和Ala-Glu-Glu(IC50:12.42μmol/L)。     

Angiotensin I-converting enzyme inhibitory properties of whey protein digests: concentration and characterization of active peptides

The aim of this study was to identify whey-derived peptides with angiotensin I-converting enzyme (ACE) inhibitory activity. The bovine whey proteins alpha-lactalbumin and beta-lactoglobulin were hydrolysed with pepsin, trypsin, chymotrypsin, pancreatin, elastase or carboxypeptidase alone and in combination. The total hydrolysates were fractionated in a two step ultrafiltration process, first with a 30 kDa membrane and then with a 1 kDa membrane. Inhibition of ACE was analysed spectrophotometrically. The peptides were isolated by chromatography and identified by mass and sequencing analysis. The most potent inhibitory peptides were synthesized by the 9-fluorenylmethoxycarbonyl solid phase method. Inhibition of ACE was observed after hydrolysis with trypsin alone, and with an enzyme combination containing pepsin, trypsin and chymotrypsin. Whey protein digests gave a 50% inhibition (IC50) of ACE activity at concentration ranges within 345-1733 micrograms/ml. The IC50 values for the 1-30 kDa fractions ranged from 485 to 1134 micrograms/ml and for the < 1 kDa fraction from 109 to 837 mg/ml. Several ACE-inhibitory peptides were isolated from the hydrolysates by reversed-phase chromatography, and the potencies of the purified peptide fractions had IC50 values of 77-1062 microM. The ACE-inhibitory peptides identified were alpha-lactalbumin fractions (50-52), (99-108) and (104-108) and beta-lactoglobulin fractions (22-25), (32-40), (81-83), (94-100), (106-111) and (142-146).     

Effects of heat treatment and in vitro digestion on the Angiotensin converting enzyme inhibitory activity of some legume species

In this study, potential Angiotensin Converting Enzyme (ACE) inhibitory activity of common dry beans, dry pinto beans and green lentils were determined and the effects of different heat treatment periods were investigated. In vitro gastrointestinal digestion was applied after heat treatment, and stability of the ACE inhibitory activity was determined. Crude protein extracts of legume samples showed 4.08–28.54% ACE inhibition activities. 30-min heat treatment caused a decrease in ACE inhibitory activity; however, 50-min heat treatment was observed to be beneficial for the release of ACE inhibitory peptides from the three legume species (p < 0.05). In vitro digestion process increased ACE inhibitory activities of the samples (p < 0.05), mainly green lentil digests by a factor of 10.91–23.65 against ACE in comparison with raw sample. It was observed that IC₅₀ values of common beans, pinto beans and green lentils after digestion were 0.78–0.83, 0.15–0.69 and 0.008–0.89 mg protein/mL, respectively.     

ACE inhibitory peptides and antioxidant peptides derived from in vitro digestion hydrolysate of hen egg white lysozyme

Lysozyme from hen egg white is a well-known antimicrobial protein with high ratio of hydrophobic and positively charged amino acid residues. In order to explore functional bioactivities of enzymatic hydrolysates of lysozyme, the protein was subjected to a simulated gastrointestinal digestion and the resulting hydrolysate (LPH2) showed a strong competitive angiotensin I-converting enzyme (ACE) inhibitory activity (IC(50)=12.6μg/ml) and a remarkable antioxidant activity. The LPH2 was fractionated using a 3kDa cut-off membrane and the obtained permeate LPH2-3kDa was analysed by MALDI-TOF-TOF MS. Using this technology, 38 different peptides were identified and some of these peptides were well fit with structure requirements of ACE inhibitory peptides and/or antioxidant peptides. The findings from this study suggest that the protein containing high proportion of hydrophobic and positively charged residues have the potential to generate multifunctional peptides, and these peptides would be beneficial ingredient to be used in functional foods.     

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。     

刺参ACE抑制肽制备及降压功效分析

以刺参（Stichopus japonicus）体壁为原料,利用复合蛋白酶对其进行酶解,运用单因素法优化血管紧张素转化酶（angiotensin I-converting enzyme,ACE）抑制肽的酶解制备工艺。获得最佳工艺参数为：料液比1∶6（g/mL）、加酶量0.8%（质量分数）、反应时间6?h、pH?7.0。将酶解产物用3?kDa膜超滤,其中小于3?kDa组分具有较高的ACE抑制活性,IC50为0.8?mg/mL,对ACE表现为非竞争性抑制作用。刺参ACE抑制肽在高温、酸性和碱性条件下均具有较好的稳定性,同时具有较强的抵抗胃蛋白酶、胰蛋白酶和胰凝乳蛋白酶消化的能力。动物实验结果表明,刺参ACE抑制肽对雄性自发性高血压大鼠具有良好的降压功效。因此,利用复合蛋白酶酶解刺参体壁制备的ACE抑制肽在降血压功能性食品的开发利用方面具有研究价值。     

食物源ACE抑制肽的生产及构效关系研究进展

生物活性肽是蛋白质中隐藏着的可在体内发挥一定生理功能的活性肽段。目前已从食物蛋白中获得了具有免疫调节、抑菌、抗病毒、抗肿瘤、抗血栓形成、抗高血压等功能的多肽,其中血管紧张素转化酶(ACE)抑制肽是研究热点之一。ACE通过产具有升压作用的血管紧张素Ⅱ和降解扩张血管的舒缓激肽在调节血压中发挥着重要作用。ACE抑制肽可通过消化道水解、发酵和熟化过程、体外酶解和遗传重组方法而获得,并可作为功能因子添加到功能食品中,相关产品已上市或研发中。本文对ACE抑制肽的生产及其构效关系研究进展等进行综述。     

Antihypertensive and antithrombotic activities of a commercial fermented milk product made with Lactobacillus casei Shirota and Streptococcus thermophilus

Peptides which inhibit the human angiotensin‐converting enzyme (ACE) may be released during milk fermentation, and the micro‐organisms or fermentation conditions influence the specific peptides produced. The aim of this study was to evaluate the ACE inhibitory and antithrombotic activities of a fermented milk product commercially available in Mexico. Viable cell numbers, protein hydrolysis and the pH remained constant during refrigerated storage. The IC₅₀ of ACE inhibitory activity was 31.38 mg/mL. Eight peptide fractions exhibited ACE inhibitory activity and six showed antithrombotic activity. Two fractions showed both. This is the first time that both activities have been reported in a commercial probiotic product.     

源于皱纹盘鲍裙边胶原蛋白ACE抑制肽的制备

为了提高皱纹盘鲍(Haliotis discus hannai)裙边的利用率,以其为原料纯化胶原蛋白,并通过胰蛋白酶和胃蛋白酶共酶解制备血管紧张素转换酶(angiotensin I-converting enzyme,ACE)抑制肽。酶解液经超滤、Superdex^TM peptide10/300GL凝胶柱和高效液相色谱分离纯化,获得了3条来源于皱纹盘鲍胶原蛋白的肽段SGEVGQ、QRGPAGAQGPQ和GPPGPAGAR。其中结合能力最强的肽为GPPGPAGAR,对ACE的IC50值为177.1μmol/L,分子对接结果显示其主要作用于ACE的S1活性口袋,抑制模式与赖诺普利类似,并且经模拟胃肠液消化后仍能发挥较强的ACE抑制作用。本研究通过酶解皱纹盘鲍裙边胶原蛋白制备ACE抑制肽,为鲍鱼裙边的精深加工和ACE抑制肽的开发提供了参考。