Effect of angiotensin I-converting enzyme (ACE) inhibitory peptide purified from enzymatic hydrolysates of <Emphasis Type="Italic">Styela plicata</Emphasis>

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from Styela plicata. The S. plicata was hydrolyzed with various proteases including Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, α-chymotrypsin, pepsin, and papain. The hydrolysate prepared with Protamex had the highest ACE inhibitory activity compared to the other hydrolysates. We attempted to isolate ACE inhibitory peptides from hydrolysate prepared with Protamex using ultra-filtration, gel filtration on a Sephadex G-25 column and reversed-phase high-performance liquid chromatography (RP-HPLC) on an ODS column. IC₅₀ value of the purified ACE inhibitory peptide was 24.7 μM, and Lineweaver–Burk plots suggest that the purified peptide from S. plicata acts as mixed-type inhibitor against ACE. Amino acid sequence of the purified peptide was identified as Met-Leu-Leu-Cys-Ser, with a molecular weight 566.4 Da. The results of this study suggest that peptides derived from S. plicata may be beneficial as anti-hypertension compounds in functional foods resource.     

Evaluation of angiotensin I-converting enzyme (ACE) inhibitory activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates generated by gastrointestinal proteases: identification of the most potent active peptide

In this study, smooth hound protein hydrolysates (SHPHs), obtained by treatment with various gastrointestinal proteases, were analyzed for their angiotensin I-converting enzyme (ACE) inhibitory activities. Protein hydrolysates were obtained by treatment with crude alkaline enzyme extract, low molecular weight (LMW) alkaline protease, trypsin-like protease and pepsin from Mustelus mustelus, and bovine trypsin. All hydrolysates exhibited inhibitory activity toward ACE. Hydrolysate generated with alkaline protease extract displayed the highest ACE inhibitory activity, and the higher inhibition activity (82.6% at 2 mg/mL) was obtained with a hydrolysis degree of 18.8%. This hydrolysate was then fractionated by size exclusion chromatography on a Sephadex G-25 into five major fractions (P₁–P₅). ACE inhibitory activities of all fractions were assayed, and P₃ was found to display a high ACE inhibitory activity (62.24% at 1 mg/mL). P₃ was then fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and ten fractions of ACE inhibitors were found (F₁–F₁₀). Sub-fraction F₃ showed the strongest ACE inhibitory activity, being able to suppress more than 60% of initial enzyme activity at a concentration of 100 μg/mL. The amino acid sequence of peptide F₃ was determined by ESI/MS and ESI–MS/MS as Ala-Gly-Ser, and the IC₅₀ value for ACE inhibitory activity was 0.13 ± 0.03 mg/mL. Further, purified peptide F₃ maintained inhibitory activity even after in vitro digestion with gastrointestinal proteases in order to demonstrate gastrointestinal stability digestion to enable oral application. These results indicate that smooth hound protein hydrolysate possesses potent antihypertensive activity.     

Angiotensin I‐converting enzyme inhibitory activity of protein hydrolysates prepared from three freshwater carps (Catla catla,Labeo rohita and Cirrhinus mrigala) using Flavorzyme

Fish protein hydrolysates from three freshwater carps, Catla catla, Labeo rohita and Cirrhinus mrigala with different degree of hydrolysis (DH) (5%, 10%, 15% and 20%), were prepared using Flavorzyme enzyme and designated as HCF, HRF and HMF, respectively. The angiotensin I‐converting enzyme (ACE) inhibitory activity of hydrolysates was found to vary from 43 ± 2% to 71 ± 3%. Based on ACE inhibitory activity, HRF with DH‐15% was taken up for further study. The mode of ACE activity inhibition by HRF‐DH 15% was mixed type as revealed by Lineweaver–Burk plot. Sequential digestion of HRF‐DH 15% using pepsin and pancreatin decreased the ACE inhibitory activity from 76% to 63%. Partial purification of HRF‐DH 15% by size exclusion chromatography gave three different fractions designated as F‐1, F‐2 and F‐3 with the molecular mass in the range of 6456–407 Da. Fraction 2 had significantly higher ACE inhibitory activity than the other fractions.     

Angiotensin-I-converting enzyme (ACE)-inhibitory peptides from Thai jasmine rice bran protein hydrolysates

Rice bran protein hydrolysate (<50 kDa RBPH) from Thai jasmine variety demonstrating a high Angiotensin I converting enzyme (ACE) inhibitory activity was purified and characterised. ACE inhibitory peptides were obtained from a two-step purification process: gel filtration and preparative reverse-phase high-performance chromatography (RP-HPLC) and then identified by mass spectrometer hybrid quadrupole-time-of-flight. A novel peptide GSGYF in the RBPH was firstly identified and found to have a partial sequence homology of Oryza sativa Japonica Group. This sequence was further synthesised to exhibit as good an inhibition potency with IC₅₀ value of 2.11 µg mL⁻¹ as Captopril (1.15 µg mL⁻¹). The cytotoxicity test revealed that this RBPH is non-toxic against Vero cells. In addition, the <50 kDa RBPH was resistant to in vitro digestion by pepsin and trypsin. These findings suggest that the RBPH containing ACE inhibitory peptides is likely to be safer and healthier than synthetic drugs and can be an effective food supplement for lowering blood pressure.     

Separation of angiotensin I‐converting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature,pH and digestive enzymes

Bovine collagen was isolated from connective tissue, a by‐product in the meat processing industry and characterised by SDS‐PAGE. Alcalase and papain were employed to generate collagen hydrolysates with different degree of hydrolysis (DH). In vitro angiotensin I‐converting enzyme (ACE) inhibitory activities were evaluated and the two most potent hydrolysates from each enzyme were separated by two‐step purification. Both alcalase‐catalysed and papain‐catalysed hydrolysates exhibited strong ACE inhibitory capacities with IC₅₀ values of 0.17 and 0.35 mg mL^?1, respectively. Purification by ion‐exchange chromatography and gel filtration chromatography revealed higher ACE inhibitory activities in one fraction from each enzyme with IC₅₀ values of 3.95 and 7.29 μg mL^?1. These peptide fractions were characterised as 6‐12 amino acid residues by MALDI‐TOF/MS. The peptides retained their activity (>90%) after exposure to processing temperature and pH and in vitro simulated gastrointestinal digestion. The present results demonstrated that collagen peptides can be utilised for developing high value‐added ingredients, for example ACE inhibitory peptides.     

Preparation and identification of angiotensin I-converting enzyme inhibitory peptides from sweet potato protein by enzymatic hydrolysis under high hydrostatic pressure

Sweet potato protein hydrolysates (SPPH) with angiotensin I-converting enzyme (ACE) inhibitory activity were prepared by papain, pepsin and alcalase under high hydrostatic pressure (HHP, 100–300 MPa). HHP significantly increased degree of hydrolysis (DH), nitrogen recovery (NR) and molecular weight (MW) <3 kDa fractions contents of SPPH by all three enzymes (P < 0.05). MW < 3 kDa peptide fractions from SPPH by alcalase under 100 MPa showed the highest ACE inhibitory activity (IC₅₀ value 32.24 µg mL⁻¹), and was subjected to purification and identification by semi-preparative RP-HPLC and LC-MS/MS. Fifty-four peptides ranged from 501.28 to 1958.88 Da with 5–18 amino acids were identified and matched sporamin A and B sequences. Five identified peptides with sequences of VSAIW, AIWGA, FVIKP, VVMPSTF and FHDPMLR displayed good ACE inhibitory activity with the contribution of Val, Trp, Phe and Arg. Thus, SPPH by enzymatic hydrolysis under HHP can be potentially used in functional food.     

Identification of novel angiotensin I-converting enzyme inhibitory peptide from collagen hydrolysates and its molecular inhibitory mechanism

The fish collagen protein was hydrolysed and further fractionated into four molecular weight ranges by ultrafiltration. Subsequently, the peptide fraction with the potent angiotensin I-converting enzyme (ACE) inhibitory activity was identified. The potential inhibitory mechanism of the peptide was clarified by molecular docking. As a result, FCPH-Ⅳ with molecular weight between 600 and 1000 Da exerted the high ACE inhibitory activity and was identified by de novo peptide sequencing. The peptide GHVGAAGS exhibited significant ACE inhibitory activity with the IC₅₀ value of 407.28 ± 3.55 μm . In addition, the docking results showed the interactions between the amino acids at the four positions closest to the C-terminal site of GHVGAAGS and the major active residues (GLN281, HIS353, LYS511 and HIS513) of ACE lead to the conformational change in ACE. This work indicates that fish collagen could be utilised to produce ACE inhibitory peptides and develop health products.     

Preparation and purification of angiotensin‐converting enzyme inhibitory peptides from hydrolysate of shrimp (Litopenaeus vannamei) shell waste

Angiotensin I‐converting enzyme (ACE) inhibitory peptides from the shrimp shell waste (SSW) were isolated using different proteases. The orthogonal test results showed alcalase hydrolysates with ACE inhibitory activity of 67.07% under the optimal hydrolysis conditions of 60 °C hydrolysis temperature, pH = 9.5, 25 g L^?1 substrate and 4000 U g^?1 of enzyme, whereas neutral protease hydrolysates had an ACE inhibitory activity of 84.04% under the hydrolysis temperature of 50 °C at pH = 7.0 with 25 g L^?1 of substrate and in the presence of 2000 U g^?1 of enzyme. Neutral protease was more suitable for the production of ACE inhibitory peptides from SSW, where peptides with MW <5 kDa were recommended. The results of this study indicated that peptides obtained from SSW are as beneficial as antihypertension compounds in the functional food resources.     

Effects of fermentation conditions on the potential anti‐hypertensive peptides released from yogurt fermented by Lactobacillus helveticus and Flavourzyme®

This study investigates the effects of fermentation conditions on the production of angiotensin‐converting enzyme inhibitory (ACE‐I) peptides in yogurt by Lactobacillus helveticus 881315 (L. helveticus) in the presence or absence of Flavourzyme^®, which is derived from a mould, Aspergillus oryzae and used for protein hydrolysis in various industrial applications. Optimal conditions for peptides with the highest ACE‐I activity were 4% (v/w) inoculum size for 8 h without Flavourzyme^® supplementation, and 1% inoculum size for 12 h when combined with Flavourzyme^®. The yogurt fermented by L. helveticus resulted in IC₅₀ values (concentration of inhibitor required to inhibit 50% of ACE activity under the assayed conditions) of 1.47 ± 0.04 and 16.91 ± 0.25 mg mL^?1 with and without Flavourzyme^® respectively. Seven fractions of ACE‐I peptides from the yogurt incorporated with L. helveticus and Flavourzyme^® were separated using the preparative high‐performance liquid chromatography. Fraction (F3) showed the highest ACE‐I activity with an IC₅₀ of 35.75 ± 5.48 μg mL^?1. This study indicates that yogurt may be a valuable source of ACE‐I peptides, which may explain the outcomes observed in the experimental and clinical studies and foresee the application of fermented milk proteins into functional foods or dietary supplements.     

Selective separation and characterisation of dual ACE and DPP-IV inhibitory peptides from rainbow trout (Oncorhynchus mykiss) protein hydrolysates

Microwave pretreatment and hydrolysis were applied to rainbow trout (Oncorhynchus mykiss) by-products to produce bioactive peptides with dual in vitro angiotensin-I converting enzyme (ACE) and dipeptidyl-peptidase IV (DPP-IV) inhibitory activities. Peptides were fractionated using the single step electrodialysis with ultrafiltration membrane (EDUF). Concentration of cationic peptides (CP) increased in the recovery solution, reaching 125 μg mL⁻¹ after a 4-h treatment with migration rate of 15.68 ± 2.98 g m⁻² h. CP fractions displayed ACE and DPP-IV I inhibitory properties, with IC₅₀ values of 0.0036 mg mL⁻¹ and 1.23 mg mL⁻¹ respectively. The bioactivity was attributed to the low molecular weight peptides (300–500 Da) recovered. CP exhibited non-competitive inhibition patterns for ACE and DPP-IV, which were dose dependent. These results showed that bioactive peptides can successfully be separated from complex hydrolysate mixtures by EDUF. The fractionated peptides can serve as potential functional food ingredients or nutraceuticals for the management of hypertension and diabetes.     

Optimization of the Antibacterial Activity of Half-Fin Anchovy (<Emphasis Type="Italic">Setipinna taty</Emphasis>) Hydrolysates

In some cases, food proteins hydrolyzed by protease can release antibacterial peptides. In this study, antibacterial activities of half-fin anchovy (Setipinna taty), digested by papain, pepsin, trypsin, alkaline protease, acidic protease, and flavoring protease, were measured, respectively. Additionally, the mechanism of antibacterial action was investigated. Results showed that half-fin anchovy pepsin hydrolysate displayed higher antibacterial activity than other hydrolysates. Response surface methodology was then used to optimize pepsin hydrolysis parameters using a central composite design method. The results demonstrated that pepsin-to-substrate level of 1,100 U/g, pH of 2.0, reaction time of 2.4 h, and water-to-substrate ratio of 4:1 (v/w) were the optimal conditions to generate antibacterial hydrolysate. The optimized half-fin anchovy pepsin hydrolysate (HAHp) effectively inhibited the growth of Escherichia coli CGMCC 1.1100, Pseudomonas fluorescens CICC 20225, Proteus vulgaris CICC 20049, and Bacillus megaterium CICC 10324 with the minimal inhibitory concentration values ranging from 28.38 to 56.75 μg/ml. The cell integrity of E. coli CGMCC 1.1100 was significantly destroyed after incubation with HAHp for 5 h (p < 0.05), and cell membrane damage was also observed in scanning electron microscopy. It could be concluded that the antibacterial mechanism was partially due to the ability of HAHp to destroy bacterial cell integrity via irreversible cell membrane damage. Moreover, amino acid composition analysis showed that HAHp’s nutritional value was superior to the reference used by WHO/FAO, indicating that HAHp could be used as a functional antibacterial agent in food or feed.     

α 67-106 of bovine hemoglobin: a new family of antimicrobial and angiotensin I-converting enzyme inhibitory peptides

Protein hydrolysates are of a significant interest, due to their potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. The present study was focused on bovine hemoglobin hydrolysate obtained with pig pepsin in the presence of 30% ethanol. This hydrolysate was fractioned by reversed-phase high-performance liquid chromatography (RP-HPLC) into 12 major fractions (F₁–F₁₂). All fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize and identify the peptides in these fractions. This hydrolysis permitted to generate a new serial of bioactive peptides with both antimicrobial and ACE inhibitory activities. Identified peptides were TKAVEHLDDLPGALSELSDLHAHKLRVDPVNFKLLSHSLL, LDDLPGALSELSDLHAHKLRVDPVNFKLLSHSL, KLLSHSL, and LLSHSL corresponding respectively to the 67-106, 73-105, 99-105, and 100-105 fragments of the α chain of bovine hemoglobin. They were the first found from bovine hemoglobin. These purified peptides have an antibacterial activity against four bacteria strains: Kocuria luteus A270, Listeria innocua, Escherichia coli, and Staphylococcus aureus with a MIC between 187.1 and 35.2 μM. On the other hand, these peptides displayed at the same time ACE inhibitory activity with an IC₅₀ range from 42.55 to 1,095 μM.     

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。     

酶解猪血浆蛋白粉制备ACE抑制肽的工艺优化

选用胃蛋白酶、胰蛋白酶、碱性蛋白酶和中性蛋白酶水解猪血浆蛋白粉,通过单因素和正交试验考察其水解产物对ACE的抑制活性,并优选酶解工艺条件。结果显示:胃蛋白酶适宜水解血浆蛋白粉,制备ACE抑制肽;影响胃蛋白酶水解的4个因素主次顺序为底物质量浓度>酶解时间>pH值>酶与底物质量比(m酶:m底物),其中底物质量浓度和酶解时间的影响显著(P<0.05),pH值和m酶:m底物的影响不显著(P>0.05);适宜胃蛋白酶水解的条件为pH2.3、水解时间1.5h、底物质量浓度1g/100mL、m酶:m底物1:6。     

乳清蛋白酶解制备ACE抑制肽的研究

采用碱性蛋白酶、中性蛋白酶、胃蛋白酶、胰蛋白酶和木瓜蛋白酶水解乳清蛋白制备ACE抑制肽，通过体外检测法测定其ACE抑制率。结果表明，碱性蛋白酶水解物的ACE抑制率最大。采用三因素二次通用旋转设计对碱性蛋白酶水解乳清蛋白的水解条件进行优化。研究了底物浓度、温度和酶与底物的质量比对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 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).     

分步酶解酪蛋白制备小分子ACE抑制肽

通过模拟胃肠道消化,采用单酶和复合酶分步水解酪蛋白获得小分子的血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制肽。首先通过胃蛋白酶水解条件的优化获得具有高ACE抑制活性肽。然后以此为底物通过胰蛋白酶和胰凝乳蛋白复合酶水解条件优化获得具有高ACE抑制活性的小分子肽。结果表明：第一步的胃蛋白酶水解最优条件为：[E]/[S]=6%、[S]=0.015g/mL、pH=1.8、t=37℃、t=2h,水解产物稀释10倍后ACE抑制率为84.5%,分子质量集中在2000D以下;第二步的复合酶水解最优条件为：m胰蛋白酶(6%):m胰凝乳蛋白酶(3%)=2:1、pH=7.8、t=48℃、t=5h,水解产物稀释10倍后ACE抑制率为85.9%,分子质量集中在500D以下。研究表明,通过分步酶解选择合适的酶解条件可以获具有较高ACE抑制活性的小分子肽。     

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.     

Effect of enzyme type and hydrolysis conditions on the in vitro angiotensin I‐converting enzyme inhibitory activity and ash content of hydrolysed whey protein isolate

Removal of salts from protein hydrolysate mixture on large scale is very difficult and relatively inefficient. Selecting practical proteinase system and hydrolysis conditions for the production of whey protein isolate (WPI) enzymatic hydrolysates with high angiotensin I‐converting enzyme (ACE) inhibitory activity and low ash content is very useful. The effect of alcalase, neutrase, trypsin and their combined system, i.e. alcalase‐neutrase and trypsin‐neutrase, under two different hydrolysis conditions, i.e. pH‐controlled and pH‐spontaneous drop, on the formation of ACE‐inhibitory peptides and the characteristics of WPI hydrolysate was investigated. Results showed that the ACE‐inhibitory activity of WPI hydrolysate obtained with alcalase was significantly higher than that of its trypsin or neutrase hydrolysate obtained at the same hydrolysis time by both pH‐controlled and pH‐spontaneous drop method (P < 0.05). The WPI hydrolysate obtained after 3 h incubation with alcalase plus 2 h with neutrase under pH‐spontaneous drop condition possessed the highest ACE‐inhibitory activity of 54.30% and the lowest ash content of 2.95%. This is practical as a functional ingredient in the food industry because of its high ACE‐inhibitory capability, commercial availability in large supply of alcalase and neutrase and no needing for additional desalting process.     

菠萝蛋白酶水解泥鳅蛋白制备ACE抑制肽的研究

为了探讨利用泥鳅蛋白制备功能性肽的可能性,采用高效液相色谱法测定泥鳅肉水解物对血管紧张素转换酶(ACE)的抑制作用,从胰蛋白酶、胃蛋白酶、菠萝蛋白酶、复合风味蛋白酶、复合蛋白酶5种酶中筛选出菠萝蛋白酶作为酶解泥鳅肉制备具有降血压活性水解物的适宜水解酶。在单因素试验的基础上,采用L9(34)正交试验设计对该酶的酶解条件进行优化。结果表明最佳水解条件为：温度55℃,固液比1:3,pH6.5,加酶量1000U/g pro,水解时间90min。在该条件下,水解物的ACE抑制率IC50值为0.0184mg/mL,ACE抑制肽的相对分子质量主要集中在924左右。