Characterization of ACE-inhibitory peptide associated with antioxidant and anticoagulation properties

A bioactive peptide Arg-Val-Pro-Ser-Leu (RVPSL) obtained from egg white protein was characterized by LC-MS and further chemically synthesized by the Fmoc solid phase method and investigated in terms of its angiotensin converting enzyme (ACE)-inhibitory activity, antioxidant property, and anticoagulation activity, as well as its stability in a simulated gastrointestinal digestion. The peptide exhibited an ACE-inhibitory activity with an IC(50) value of 20 μM. Also, the peptide could efficiently quench the (1,1)-diphenyl-2-picrylhydrazyl free radicals and exhibit high anticoagulation activity with a complete inhibition at 100 mM. Moreover, the peptide has a good stability against protease digestion. These results suggest that the peptide RVPSL may have potential to be used in nutraceuticals and functional food. Practical Application: The present research revealed a novel multifunctional peptide hydrolyzed from egg white protein. The peptide RVPSL was not only able to block the amplification of the coagulation cascade, but also able to inhibit ACE activity.     

Angiotensin I-converting enzyme-inhibitory peptide fractions from albumin 1 and globulin as obtained of amaranth grain

A number of biopeptides promoting health benefits have been isolated from food-protein hydrolysates and can be released during enzymatic digestion. Antihypertensive peptides can be part of protein fractions from amaranth grain. The objective of this work was to obtain ACE-inhibitory peptide fractions from albumin 1 and the globulin of amaranth (Amaranthus hypochondriacus) grain. Albumin 1 and globulin were hydrolysed with alcalase; hydrolysis was monitored by proteolytic degradation and by ACE-inhibitory activity. The highest ACE-inhibitory activity was 40% and 35% as obtained after 18 and 15 h hydrolysis for albumin 1 and globulin, respectively. Further separation and purification of the ACE-inhibitory peptide fractions were carried out by gel filtration and C₁₈ RP–HPLC. The IC₅₀ was 0.35 ± 0.02 mg/ml for albumin 1 peptide fraction and 0.15 ± 0.03 mg/ml for globulin peptide fraction. Albumin 1 peptide fraction showed an competitive mode of ACE inhibition, whereas the globulin peptide fraction was competitive. The globulin peptide fraction may have one of the most active naturally-occurring ACE-inhibitory peptides.     

In vivo anti-hypertensive effect of peptides from egg white and its molecular mechanism with ACE

Previous work has demonstrated that peptides QIGLF and RVPSL exhibited in vitro ACE inhibitory activity. This study was aimed to evaluate the in vivo anti-hypertensive effects of peptides RVPSL and QIGLF using animal experiments, and clarify the molecular mechanisms of interaction between ACE and peptides by molecular docking. In this work, the systolic blood pressure (SBP) of SHRs treated with QIGLF and RVPSL decreased by 48 ± 6 and 46 ± 6 mmHg, respectively. Docking results revealed that QIGLF and RVPSL established interactions with three main actives that of ACE, that is, S1 (Ala 354), S2 (Gln 281, His 513, Tyr 520 and Lys 511) and S1’ (Glu 162). These interactions can prevent ACE from binding to substrate and competitive inhibition. The in vivo anti-hypertensive effect of QIGLF and RVPSL was consistent with their in vitro ACE inhibitory activity. QIGLF and RVPSL have potential to be a healthy functional food with anti-hypertensive effects.     

Angiotensin I-converting enzyme inhibitory activity of enzymatic hydrolysates of goat milk protein fractions

Casein and whey protein fractions from goat milk were hydrolysed by subtilisin and trypsin, individually and in combination, to release angiotensin converting enzyme (ACE)-inhibitory peptides. Selected hydrolysates were fractionated by size exclusion chromatography (SEC) and further characterised. The highest ACE-inhibitory activity was obtained from the casein fraction hydrolysed by the combination of enzymes. SEC presented 4 fractions with fraction F2 (<2.3 kDa) containing the highest concentration of peptides and the highest activity. F2 contained a number of peptides not previously identified from caprine caseins but with structural similarity to other ACE-inhibitory peptides. The most active fraction in relation to protein content was F4 with IC₅₀ between 9.3 and 5.1 μg mL⁻¹. This fraction contained a compound tentatively identified as WY, an active dipeptide not previously reported from caseins. The high inhibitory capacity of these fractions points towards the advantage of implementing a membrane process to concentrate the most active peptides.     

Angiotensin I-converting enzyme inhibitory properties and SDS-PAGE of red lentil protein hydrolysates

Several research studies have shown that protein hydrolysates from milk and soy contain peptides that possess angiotensin I converting enzyme (ACE) inhibitory properties and may help to prevent hypertension. To date, no studies have been conducted to determine if red lentil (Lens culinaris) proteins contain peptides with ACE-inhibitory properties. The objective of the present work was to characterize the proteins present in red lentils and determine if tryptic hydrolysis could liberate peptides with ACE-inhibitory properties. Red lentil protein extracts were prepared and fractionated to obtain enriched albumin, legumin and vicilin fractions. Protein/peptide profiles were studied by electrophoresis and ACE-inhibitory activity was measured using the HPLC hippuryl-His-Leu (HHL) substrate method. Our results revealed that red lentil protein hydrolysates posses ACE-inhibitory properties. Furthermore, we demonstrated that the ACE-inhibitory property of the hydrolysates varied as a function of the protein fraction with the total lentil protein hydrolysate having the lowest half maximal inhibitory concentration (IC₅₀) (111 ± 1 μmol/L) (i.e., highest ACE-inhibitory activity), followed by the enriched legumin (119 ± 0.5 μmol/L), albumin (127 ± 2 μmol/L) and vicilin (135 ± 2 μmol/L) fractions, respectively.     

Angiotensin I-Converting Enzyme Inhibitory Activity of Soy Protein Digests in a Dynamic Model System Simulating the Upper Gastrointestinal Tract

ABSTRACT: The objective of this study was to investigate whether peptides with inhibitory activity against angiotensin I-converting enzyme (ACE) would be produced by digestion of isolated soy protein (ISP) in a dynamic model system simulating gastrointestinal conditions. Using the model system, 5% ISP solution was pumped into the stomach reactor containing pepsin and HCl. The peptic digest was continuously pumped into the duodenum reactor containing pancreatin and Oxgall bile. The effects of blanching (100°C, 10 min) followed by pasteurization (75°C, 15 s) or sterilization (121°C, 20 min) of ISP before digestion on the inhibitory activity were also investigated. During the first 30 min of digestion, significantly higher ( P < 0.05) ACE-inhibitory activity was generated from unheated ISP after sequential digestion in both reactors compared with after peptic digestion only in the stomach reactor. However, at 90 min, subsequent digestion by pancreatin of unheated and blanched-sterilized ISP decreased ACE-inhibitory activity compared with peptic digestion alone. The IC₅₀ values at the end of 90 min digestion in both reactors were 0.38 ±0.01, 0.37 ± 0.02, and 0.44 ± 0.02 mg/mL for unheated, blanched-pasteurized, and blanched-sterilized ISP, respectively. The results suggest the potential production of peptides with ACE-inhibitory activity upon physiological digestion of soy protein, including products that have been subjected to heat processing. Although clinical trials would be required to provide final evidence of efficacy of the soy peptides, the present findings support the application of soy protein as an ingredient for functional foods.     

Activity and bioavailability of food protein-derived angiotensin-I-converting enzyme–inhibitory peptides

Angiotensin-I-converting enzyme (ACE) inhibitory peptides are able to inhibit the activity of ACE, which is the key enzymatic factor mediating systemic hypertension. ACE-inhibitory peptides can be obtained from edible proteins and have the function of antihypertension. The amino acid sequences and the secondary structures of ACE-inhibitory peptides determine the inhibitory activities and stability. The resistance of ACE-inhibitory peptides to digestive enzymes and peptidase affect their antihypertensive bioactivity in vivo. In this paper, the mechanism of ACE-inhibition, sources of the inhibitory peptides, structure–activity relationships, stability during digestion, absorption and transportation of ACE-inhibitory peptides, and consumption of ACE-inhibitory peptides are reviewed, which provide guidance to the development of new functional foods and production of antihypertensive nutraceuticals and pharmaceuticals.     

Potential ACE-inhibitory activity and nanoLC-MS/MS sequencing of peptides derived from aflatoxin contaminated peanut meal

Our lab has developed a process for sequestering aflatoxin from contaminated peanut meal (PM) using commercial bentonite clays while protein is simultaneously extracted and hydrolyzed by a commercial protease. The objectives of this study were to sequence generated peptides and evaluate their potential ACE-inhibitory properties. Aflatoxin in the unprocessed PM was 610 μg kg⁻¹ compared to 9.7 μg kg⁻¹ on a dry weight basis in the 120 min hydrolysate. This hydrolysate displayed significant ACE-inhibitory activity with an IC₅₀ of 295.1 μg mL⁻¹. Ultrafiltration and size exclusion chromatography (SEC) improved the ACE-inhibitory properties, with the SEC fraction containing the smallest peptides having an IC₅₀ = 44.4 μg mL⁻¹. Additionally, 271 unique peptides were identified by nanoLC-MS/MS, of which 147 belonged to major seed storage proteins. This advanced characterization data will ultimately allow for more efficient production of hydrolysates with ACE-inhibitory activity or other bioactivities of interest from PM.     

Antihypertensive,ACE-inhibitory and vasodilator properties of an egg white hydrolysate: Effect of a simulated intestinal digestion

In previous studies we showed that the egg white hydrolysate with pepsin (HEW) and its fraction with molecular mass lower than 3000 Da (HEW < 3000) have ACE-inhibitory activity in vitro and exert antihypertensive effects after single-oral and long-term administrations to spontaneously hypertensive rats (SHR). In this paper we have simulated an intestinal digestion on HEW and HEW < 3000 and addressed its effect on their ACE-inhibitory and vasodilator activities in vitro and antihypertensive activity in vivo. The results showed that the ACE-inhibitory activity of HEW and HEW < 3000 was maintained after the simulated intestinal digestion. HEW also exerted vasodilator activity in isolated aortic rings before and after the digestion. Both activities may explain the antihypertensive effects of these products in SHR. The peptides RADHP and YPI, which were detected by RP–HPLC–MS in the hydrolysates after the action of the pancreatic enzymes, could be responsible for the antihypertensive effects. In conclusion, HEW or HEW < 3000 could be useful in the prevention and/or treatment of hypertension and other associated disorders.     

Production of whey protein isolate hydrolysate fractions with enriched ACE-inhibitory activity

A study on the enrichment of angiotensin-converting enzyme (ACE) inhibitory activity in whey protein isolate (WPI) hydrolysate fractions is presented. A previously identified low molecular mass fraction (1 kDa permeate) of an enzymatically hydrolysed heat-treated WPI with elevated ACE-inhibition (IC₅₀ = 0.23 g L⁻¹) was subjected to cascade membrane ultrafiltration (UF) and diafiltration steps at lab-scale. Assaying for ACE-inhibition revealed that the 1 kDa retentate demonstrated the highest ACE-inhibitory activity (IC₅₀ = 0.17 g L⁻¹). Isoelectric focussing (IEF) of the hydrolysate fraction further increased ACE-inhibition in fractions collected within the pH range 6.1–6.6. Overall, both UF and IEF enriched the ACE inhibitory activity in the original fraction by ∼52%, demonstrating the potential for enrichment of bio-functional activities in enzymatic hydrolysates of whey proteins.     

Angiotensin I-converting (ACE)-inhibitory and anti-inflammatory properties of commercially available Greek yoghurt made from bovine or ovine milk: A comparative study

The objective of this study was to compare the presence of peptides with angiotensin I-converting (ACE)-inhibitory and immunomodulatory activities in commercial Greek yoghurt made from bovine or ovine milk. Water soluble extracts (WSE) in yoghurts made from ovine milk exhibited higher ACE-inhibitory activity than their bovine counterparts. Similarly, ovine yoghurt WSE inhibited more effectively the expression of two pro-inflammatory genes (inducible NO synthase and cyclooxygenase-2) by ovine monocytes. Following gel filtration of WSE by Sephadex G25, one fraction exhibited only ACE-inhibitory activity, two only immune inhibitory while one possessed both activities. Thus, selected peptidic fractions mimicked the original effects observed in WSE prior to fractionation.     

Modification of Soybean Protein Hydrolysates by Alcalase-Catalyzed Plastein Reaction and the ACE-Inhibitory Activity of the Modified Product In Vitro

Soybean protein hydrolysates were prepared by hydrolyzing soybean protein isolates with a protease alcalase to a degree of hydrolysis of 16.6%, and then modified by alcalase-catalyzed plastein reaction to reveal the impact of plastein reaction on the ACE-inhibitory activity of the modified product in vitro. The suitable conditions of plastein reaction of soybean protein hydrolysates were selected based on the results of response surface methodology with the decreased amount of the free amino groups of the modified product as response. When reaction temperature was fixed at 30°C, the selected conditions were as follows: concentration of soybean protein hydrolysates of 45% (w/w), addition level of alcalase of 275 U/g peptides, and reaction time of 3 to 4 h. Soybean protein hydrolysates and eight modified products were evaluated for their ACE-inhibitory activities in vitro. The assay results highlighted that plastein reaction improved the ACE-inhibitory activity of the modified product. The IC₅₀ of the modified products ranged from 0.64 to 1.11 mg/mL, while that of soybean protein hydrolysates was 1.45 mg/mL. The decreased amount of the free amino groups of the modified product showed influence on the ACE-inhibitory activity in vitro. Analysis results from size exclusion chromatography confirmed that some plasteins with higher molecular weights were formed in the modified product. Our results showed that alcalase-catalyzed plastein reaction could be applied as a potential approach to enhance the ACE-inhibitory activity of soybean protein hydrolysates in vitro.     

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).     

ACE-inhibitory and antioxidant properties of potato (Solanum tuberosum)

Proteins were isolated from potato tubers (Solanum tuberosum) at different physiological states, and by-products from the potato industry were used to evaluate their ACE-inhibitory and radical-scavenging potencies. Protein isolates and by-products were autolysed or hydrolysed by alcalase, neutrase and esperase. Hydrolysis increased the inhibition of the angiotensin-converting enzyme (ACE) and the radical-scavenging activity. The ACE-inhibitory potencies of the hydrolysates were high (IC₅₀ = 0.018 − 0.086) and the by-product fractions showed ACE-inhibition also before hydrolysis. All samples exhibited low radical-scavenging activity, and hydrolysis for 2 h with proteases was needed to produce an increase in the activity. Ultrafiltration through 10–3 kDa membranes efficiently separated the ACE-inhibitory compounds into permeate fractions. The results of this study suggest that potato is a promising source for the production of bioactive compounds as ingredients for developing functional foods with a beneficial impact on cardiovascular health.     

超声波-离子液体耦合预处理制备蚕蛹蛋白质ACE抑制肽的工艺优化

为提高蚕蛹蛋白质酶解产物的ACE抑制活性,利用超声波-离子液体耦合法对蚕蛹蛋白质进行预处理。以酶解产物ACE抑制活性为指标,采用单因素结合响应面分析法,研究超声波-离子液体耦合预处理蚕蛹蛋白质的工艺条件,并通过SDS-聚丙烯酰胺凝胶电泳,研究预处理前后蚕蛹蛋白质及其酶解产物相对分子质量的变化。结果表明,各因素对酶解产物ACE抑制活性的影响程度由大到小依次为:液料比、超声波功率、预处理时间。确定最佳预处理工艺条件为:液料比27.2 mL/g,处理时间31.9 min,超声波功率406.8 W。在此优化条件下,蚕蛹蛋白质酶解产物的ACE抑制率为75.7%(IC50=0.071 mg/mL)。与未处理、超声波预处理的相比,超声波-离子液体耦合预处理在制备蚕蛹蛋白质ACE抑制肽上具有明显优势。超声波-离子液体耦合预处理后,蚕蛹蛋白质的分子质量无明显变化,但其酶解产物的分子质量(1.43 ku)变小。     

坛紫菜降血压活性肽的分离纯化及分子质量测定

用AS.1398 中性蛋白酶酶解制备坛紫菜(Porphyra haitanesis)ACE 抑制肽,并经超滤膜、Sephadex G-15 凝胶层析和反相高效液相色谱(RP-HPLC)分离纯化,纯化产物测定其氨基酸组成,并运用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)测定分子质量。结果显示：坛紫菜酶解液经超滤分离获得分子质量小于2000D 的组分ACE 抑制活性最高,IC50 为0.73mg/mL;该组分进行Sephadex G-15 凝胶层析分离得6 个组分,组分E 的IC50 最小,为0.67mg/mL。组分E 经RP-HPLC 分离得峰6 对ACE 的抑制率最高,达到89.54%;峰6 再次过RP-HPLC,又洗出2 个蛋白峰,说明峰6 并不是单一物质,还需进行多次反复纯化。测定峰6 的氨基酸组成主要含有Tyr、Val 和Phe,同时采用MALDI-TOF-MS 分析发现有8 个主要的质子峰,信号最强的质子峰为m/z 861.17,有4 个峰聚集在m/z 860 左右,说明峰6 平均分子质量大约为860D,推算出紫菜降血压肽含有3 个Val、2 个Phe、1 个Tyr,N 端为Val,可能的排序顺序有18 种。     

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.     

Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate

Squid gelatin obtained from inner and outer tunics was hydrolysed with Alcalase to isolate antioxidant peptide sequences. The ACE-inhibitory activity of the isolated peptides was also evaluated. After fractionation by ultrafiltration and size-exclusion chromatography into four fractions, the antioxidant activity of the peptide fractions was determined by radical scavenging ability and ferric reducing power. Fraction FIII showed the highest antioxidant activity, although slight differences could be expected in the antioxidant activity of the different fractions based on the amino acid composition. FIII was subjected to liquid chromatography and tandem mass spectrometry (LC–MS/MS) and two major compounds were identified: the compound with m/z 952.42, which could be mostly comprised by the carbohydrate fucose, and the peptide with m/z 1410.63. Three possible sequences were proposed and synthesised for this peptide, and the contribution of Leu or Hyp residues to the antioxidant and ACE-inhibitory activities of the resulting sequence was evaluated. The presence of Leu residues in the peptide sequence in replacement of Hyp seems to play an important role in the antioxidant and ACE-inhibitory activity.     

Angiotensin I-converting enzyme inhibitory activity in milk fermented by wild-type and peptidase-deletion derivatives of Lactobacillus helveticus CNRZ32

Development of molecular methods has enabled detailed studies of the proteolytic system of Lactobacillus helveticus, which has a central role in the release of bioactive peptides during the fermentation of milk. The impact of general aminopeptidase (PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) activity of L. helveticus on the level of angiotensin I-converting enzyme (ACE) inhibitory activity in fermented milk was elucidated by taking advantage of peptidase-negative derivatives of L. helveticus CNRZ32. According to the results, increased level of ACE-inhibitory activity was attained in milk fermented by the peptidase-deficient mutants. The ACE-inhibitory activity determined with the PepN-deficient strain decreased towards the end of fermentation, suggesting that PepN gives rise to a provisional accumulation of ACE-inhibitory peptides before their hydrolysis to shorter peptides or free amino acids. The ACE-inhibitory activity determined with the PepX-deficient strain increased throughout the fermentation, indicating specific blocking of the further hydrolysis of ACE-inhibitory peptides.     

Novel peptides from dried squid head by-products obtained from snack process

The novel peptides were successfully produced with commercial protease hydrolysis (Alcalase® and Flavourzyme®) at the fixed concentration of 3%by weight of protein in substrate. The hydrolysis was performed at various hydrolysis times (0, 300, 450, 600 and 750 min) at the optimal pH and temperature. When the hydrolysis was performed until 750 min, the peptide solutions with high soluble protein content were resulted (47.58 and 42.53 mg protein per mL for Alcalase® and Flavourzyme® hydrolysis, respectively). After freeze drying, the peptide powder from Alcalase® and Flavourzyme® hydrolysis had degree of hydrolysis at 71.87% and 66.39%, respectively. Alcalase® hydrolysis provided the novel peptide fraction with DPPH scavenging, ABTS activity and ACE-inhibitory activity at 64.20%, 102.50 µg mL⁻¹ and 56.98%, respectively. This peptide fraction possessed the lowest molecular weight (m/z 807.418) as detected by MALDI-TOF/MS, and the most potent peptide sequence was Arg-Glu-Gly-Tyr-Phe-Lys which has the potential as functional ingredient in novel food, snacks and beverages.