Angiotensin I-converting enzyme inhibitory activity of low-molecular-weight peptides from Atlantic salmon (Salmo salar L.) skin

Collagen extracted from Atlantic salmon (Salmo salar L.) skin (which is normally discarded in the process of manufacture) was hydrolyzed with Alcalase and papain, and treated by multistage separation. The salmon skin collagen peptides (SSCP) obtained had high protein content (91.20 ± 1.03%) and low molecular weights, 90.79% of which were less than 1000 Da. SSCP was then separated by reversed-phase high performance liquid chromatography. Eleven major fractions were collected and their angiotensin I-converting enzyme (ACE) inhibitory activity was assayed. Fractions 5 and 7 displaying higher ACE inhibitory activity were subjected to mass spectrometer to identify the ACE inhibitory peptides. A total of eleven peptide sequences were identified, and two dipeptides, Ala-Pro and Val-Arg, were selected for further ACE inhibitory activity analysis. The ACE inhibitory activities of Ala-Pro (IC₅₀ = 0.060 ± 0.001 mg/ml) and Val-Arg (IC₅₀ = 0.332 ± 0.005 mg/ml) were found to be approximately 20- and 4-fold higher than that of SSCP (1.165 ± 0.087 mg/ml), respectively.     

Angiotensin-I-converting enzyme (ACE)-inhibitory and antihypertensive properties of squid skin gelatin hydrolysates

Gelatin extracted from squid (Dosidicus eschrichitii Steenstrup) skin was hydrolysed with pepsin to prepare Angiotensin-I-Converting Enzyme (ACE) inhibitory peptide. The ACE-inhibitory activity was measured by spectrophotometric assay. The hydrolysate was fractionated into three ranges of molecular weight (6 kDa < HSSG-I < 10 kDa, 2 kDa < HSSG-II < 6 kDa, HSSG-III < 2 kDa) using an ultrafiltration unit. The HSSG-III showed the most potent ACE inhibitory activity in vitro with IC₅₀ of 0.33 mg/ml. Renovascular hypertensive rats (RHR) model was made with two-kidney one clip assay, and antihypertensive effects were studied in RHR treated with HSSG-III for 30 days by oral administration. Arterial blood pressure were measured respectively. The HSSG-III remarkably reduced the arterial blood pressure of RHR. These results suggested that hydrolysate of squid skin gelatin obtained by treatment with pepsin was a good source of peptides with ACE-inhibitory activity and had an antihypertensive effect by oral administration.     

Inhibition of angiotensin I-converting enzyme by wheat gliadin hydrolysates

A tryptic gliadin hydrolysate was fractionated into peptide fractions, which were assigned to either the central domain (CD) or terminal domains (TD) of gliadins. The domains were expected to contain amino acid (AA) sequences which, when released from the parent protein, inhibit the angiotensin I-converting enzyme (ACE), which plays a key role in regulating blood pressure. A proline (Pro) poor TD related fraction, containing the smallest peptides, showed the highest ACE inhibitory activity (IC₅₀ = 0.33 mg/ml). Additional peptidases were selected based on their in silico predicted ability to release ACE inhibitory peptides. Further hydrolysis of the tryptic hydrolysate fractions with thermolysin, Clarex, Alcalase and Esperase increased ACE inhibitory activities. Immobilised Ni²⁺-ion affinity chromatography (IMAC) purification of a TD related peptide fraction obtained by sequential hydrolysis with trypsin and thermolysin yielded a fraction with an IC₅₀ value of 0.02 mg/ml. This IMAC fraction was enriched in histidine and hydrophobic AA (Pro, Val, Ile, Leu and Phe).     

Angiotensin I-converting enzyme inhibitory properties of lentil protein hydrolysates: Determination of the kinetics of inhibition

ACE inhibitory activity was studied for different hydrolysates obtained from protein concentrates of two lentil varieties by in vitro gastrointestinal simulation, Alcalase/Flavourzyme, papain and bromelain. Protein/peptide profiles studied by electrophoresis and HPLC-SEC showed a rich composition of the hydrolysates in small peptides ranging in size from 0.244 to 1.06 kDa. ACE inhibitory activity was measured using the HPLC Hippuryl-His-Leu (HHL) substrate method. Significantly different (P < 0.05) IC₅₀ values ranging between 0.053 and 0.190 mg/ml were obtained for different hydrolysates. Furthermore, the inhibition mechanism investigated using Lineweaver–Burk plots revealed a non-competitive inhibition of ACE with inhibitor constants (K_i) between 0.16 and 0.46 mg/ml. These results demonstrate that hydrolysates of lentil proteins obtained by different enzymatic digestions may contain bioactive components.     

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.     

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.     

Purification and identification of angiotensin I-converting enzyme inhibitory peptide from buckwheat (Fagopyrum esculentum Moench)

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated and identified from buckwheat (Fagopyrum esculentum Moench). Buckwheat protein extract was prepared by stirring in water (pH 9.0) for 30 min, followed by centrifugation at 15,000g for 20 min. The protein extract was then filtered using an YM-10 membrane. An ACE inhibitor was purified using consecutive chromatographic methods including: ion-exchange chromatography, gel filtration chromatography, and reverse-phase high performance liquid chromatography. The ACE inhibitor was identified to be a tripeptide, Gly-Pro-Pro, having IC₅₀ value of 6.25 μg protein/ml, by protein sequencing system and electrospray-LC–mass spectrometry.     

Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor

Five different proteolytic enzymes, including Alcalase, Flavourzyme, trypsin, chymotrypsin and pepsin were employed to hydrolyze isolated soy protein (ISP) to produce the hydrolysates, respectively. The result indicated that hydrolysis of ISP for 0.5–6 h with Alcalase produced the highest ACE inhibitory activity. Therefore, Alcalase was selected for further study on optimization of hydrolysis conditions. The optimum conditions for Alcalase to hydrolyze ISP to produce the lowest IC₅₀ value were: E/S = 0.01, hydrolysis temperature = 50 °C, pH 9.0 and hydrolysis time = 6 h. Under these conditions, the IC₅₀ value of ISP was significantly reduced from 66.4 to 0.67 mg protein/ml. The lower IC₅₀ value represented the higher the ACE inhibitory activity. Moreover, several membranes with molecular weight cut-offs (MWCFs) of 1000–30,000Da were used to filter the hydrolysate. The 10 kDa permeate obtained from the treatment of the hydrolysate by 10,000 Da MWCF membrane could further reduce its IC₅₀ value from 0.668 to 0.078 mg protein/ml with a peptide recovery of 67.5%. An operation stability study showed that the membrane reactor system could maintain a steady production of ISP hydrolysate for over 8 h. The in vitro effect of gastrointestinal protease on ACE inhibitory activity of 10 kDa permeate was also investigated. The results suggested that gastrointestinal proteases have very little effect on the ACE inhibitory activity of 10 kDa permeate.     

Antioxidative and ACE inhibitory activities in enzymatic hydrolysates of the cotton leafworm, Spodoptera littoralis

The larvae of the cotton leafworm, Spodoptera littoralis, were used as a source of food proteins exerting possible biological activities. A simulated gastrointestinal digestion (IC₅₀ = 320 μg/ml) and digestion by mucosal enzymes (IC₅₀ = 211 μg/ml) reveals a significantly higher in vitro ACE inhibitory activity compared to hydrolysis using thermolysin (IC₅₀ = 1392 μg/ml) and alcalase (IC₅₀ = 827 μg/ml) as pretreatment. This indicates that the choice of enzymes to generate ACE inhibitory peptides is important. All hydrolysates were also tested for antioxidant activity using two tests: a radical scavenging test using DPPH and the ferric reducing antioxidant power (FRAP) assay, and they showed a similar antioxidant activity which was relatively low compared to the standard antioxidants BHT and vitamin C. As a conclusion, the data obtained suggest that insect protein can be used to generate hydrolysates, exerting both ACE inhibitory and antioxidant activity, which might be incorporated as multifunctional ingredient into functional foods.     

Characterisation of a new antihypertensive angiotensin I-converting enzyme inhibitory peptide from Pleurotus cornucopiae

This study describes the characterisation of a new angiotensin I-converting enzyme (ACE) inhibitory peptide from the fruiting body of Pleurotus cornucopiae which could be used as a functional food or nutraceutical compounds. After purification of the ACE inhibitor in an ultrafiltration, Sephadex G-25 column chromatography, successively C₁₈ and SCX solid-phase extraction and reverse-phase HPLC, two types of the purified ACE inhibitors with IC₅₀ values of 0.46 and 1.14 mg/ml were obtained. The two purified ACE inhibitors were analysed, showing two types of oligopeptides. The amino acid sequences of the two purified oligopeptides were found to be RLPSEFDLSAFLRA and RLSGQTIEVTSEYLFRH. The molecular mass of the purified ACE inhibitors was estimated to be 1622.85 and 2037.26 Da, respectively. Water extracts of P. cornucopiae fruiting body showed a clear antihypertensive effect on spontaneously hypertensive rats at a dosage of 600 mg/kg.     

Identification of angiotensin I-converting enzyme inhibitory peptides from koumiss, a traditional fermented mare's milk

Angiotensin I-converting enzyme (ACE) inhibitory activities in untreated koumiss and koumiss digested with ACE, pepsin, trypsinase, and chymotrypsin were compared and analyzed. Four novel ACE inhibitory peptides (P_I, P_K, P_M, and P_P) were purified using ultrafiltration and high performance liquid chromatography (HPLC). The classification study showed that these 4 peptides were of the true inhibitor type. The amino acid sequences of these peptides are YQDPRLGPTGELDPATQPIVAVHNPVIV, PKDLREN, LLLAHLL, and NHRNRMMDHVH, respectively. Their individual IC₅₀ (50% inhibitory concentration) values were as follows: 14.53 ± 0.21 μM, 9.82 ± 0.37 μM, 5.19 ± 0.18 μM, and 13.42 ± 0.17 μM. From sequence analysis, we determined that P_I was part of β-casein in mare's milk. The 3 peptides P_K, P_M, and P_P did not correspond with any known milk protein. The results suggest that koumiss is rich in ACE inhibitory peptides, and the ACE inhibitors in koumiss are of the pro-drug type or a mixture of the pro-drug type and the true inhibitor type. These results may provide evidence about the beneficial effects of koumiss, especially on cardiovascular health.     

Antioxidant effect derived from bioaccessible fractions of fruit beverages against H2O2-induced oxidative stress in Caco-2 cells

This work evaluates the effect of bioaccessible fractions from fruit beverages against oxidative stress (OS) in Caco-2 cells. A fruit beverage (grape + orange + apricot) (with/without milk and/or iron/zinc) was subjected to in vitro gastrointestinal digestion, and bioaccessible fractions were incubated with Caco-2 cell cultures. Following preincubation, OS was induced with 5 mM H₂O₂. Intracellular reactive oxygen species (ROS), mitochondrial potential (Δψ_m), mitochondrial metabolism (MTT test), intracellular reduced glutathione (GSH) and superoxide dismutase activity (SOD) were measured. The data evidenced viable cultures with increased mitochondrial metabolism and GSH-Rd activities, without alteration in SOD activity. Accordingly, more preserved mitochondrial integrity was also evidenced, allowing the action of antioxidant systems in preincubated cultures. Based on these data, we can conclude that a cytoprotective effect is derived from bioaccessible fractions of fruit beverages, though this effect failed to prevent intracellular ROS accumulation in Caco-2 cell cultures exposed to 5 mM H₂O₂.     

Antimicrobial and human cancer cell cytotoxic effect of synthetic angiotensin-converting enzyme (ACE) inhibitory peptides

Four peptides with high angiotensin-converting enzyme (ACE) inhibitory effect were separated from beef sarcoplasmic protein hydrolysates using commercial enzymes. They were identified as GFHI, DFHING, FHG, and GLSDGEWQ and their 50% inhibition concentration (IC₅₀) values against ACE were 117, 64.3, 52.9, and 50.5 μg/ml, respectively. These peptides were synthesised and further biological activities of these four peptides were measured, including antimicrobial, cytotoxic effect against cancer cells, and macrophage-stimulating effect. Peptide GLSDGEWQ showed growth inhibition on Salmonella Typhimurium, Bacillus cereus, Escherichia coli, and Listeria monocytogenes at a 100 ppm level but not on Staphylococcus aureus and Pseudomonas aeruginosa. Peptide GFHI showed higher inhibition activity on the growth of E. coli and P. aeruginosa at concentrations of 200 and 400 μg/ml. However, peptide FHG inhibited only P. aeruginosa at 200 and 400 μg/ml. The effect of separated peptides on breast cancer (MCF-7), lung cancer (A549), and stomach cancer (AGS) cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Peptide GFHI showed a slight decrease of MCF-7 cell viability in a dose dependent manner. When 400 μg/ml of peptide GFHI was applied to the AGS cell, its viability was decreased by 75%. However, peptide DFHINQ seemed to act as a nutrient to AGS cell because it increased its viability. None of the four peptides had a cytotoxic effect on A549 cells. Nitric oxide (NO) production of peptide GFHI by stimulation of macrophage was investigated at 100, 300, and 1000 μg/ml concentration. NO was not produced in all treatments. From these results it is expected that the ACE inhibitory peptides identified from beef sarcoplasmic protein hydrolysates have both antimicrobial and cancer cell cytotoxic effects.     

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.     

A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats

Tuna frame protein was hydrolysed using Alcalase, Neutrase, pepsin, papain, α-chymotrypsin and trypsin. Peptic hydrolysate exhibited the highest ACE I inhibitory activity among them and was fractionated into three ranges of molecular weight (below 1, 1–5 and 5–10 kDa) using an ultrafiltration membrane bioreactor system. The 1–5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from tuna frame protein (PTFP), which was composed of 21 amino acids, Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-Val-Ser-Asn-Trp-Ser-Pro-Pro-Lys-Try-Lys-Asp-Thr-Pro (MW: 2,482 Da, IC₅₀: 11.28 μm), was isolated. Lineweaver–Burk plots suggest that PTFP plays as a non-competitive inhibitor against ACE. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHR) also revealed that oral administration of PTFP can decrease systolic blood pressure significantly (P < 0.01). These results suggest that the PTFP would be a beneficial ingredient for nutraceuticals and pharmaceuticals against hypertension and its related diseases.     

Biologically active peptides obtained by enzymatic hydrolysis of Adzuki bean seeds

This study investigated the antioxidant and antihypertensive activities of peptides obtained from protein fractions of Adzuki bean seeds. Peptides were obtained by the use of hydrolytic enzymes in vitro under gastrointestinal conditions. A determination was made of the activity of the peptide inhibitors of the angiotensin I converting enzyme (ACE), and the antiradical and ion chelating activity of peptides from different protein fractions. The highest peptide levels after the absorption process (<7 kDa) were noted in the albumin fraction (50.69 μg/ml). Furthermore, it was found that peptides from the prolamin fraction were characterised by the highest antiradical activity and ACE inhibitory activity (IC₅₀ = 0.17 mg/ml). Peptides obtained from the globulin fraction showed the highest ability to chelate iron ions, and peptides from the glutelin fraction were characterised as being the most effective in the chelation of copper ions.     

Comparison of analytical methods to assay inhibitors of angiotensin I-converting enzyme

The linearity, precision and repeatability of visible spectrophotometric (VSP) and high-performance liquid chromatography (HPLC) methods for analysis of inhibitory activity of angiotensin I-converting enzyme (ACE) were compared by using several inhibitors and Hip-His-Leu (HHL) as substrates. IC₅₀ values (concentration at which ACE activity is inhibited by 50%) of 0.00206 ± 0.00005 μg/mL for captopril, 192 ± 4.53 μg/mL for soybean peptides, and 153 ± 4.29 μg/mL for grass carp peptides determined by the VSP method, and these values were 1.07, 1.07, 1.18 and 1.44-fold, respectively, higher than those from the HPLC method. In addition, the inhibitory constant (K_i value) of captopril was determined to be 7.09 nM and 4.94 nM using VSP and HPLC method, respectively. These results showed that the HPLC method revealed a higher level of sensitivity and precision, suitable for assaying ACE inhibition activity of antihyper-sensitive peptides. In contrast, the VSP method can simultaneously measure several samples with simple operations, suitable for analysis of ACE inhibition activity of food protein enzymatic hydrolysates.     

Angiotensin-I-converting enzyme inhibitory activity and bitterness of enzymatically-produced hydrolysates of shrimp (Pandalopsis dispar) processing byproducts investigated by Taguchi design

The importance of water-to-substrate ratio, protease type, percent enzyme and incubation time on hydrolysates produced from shrimp processing byproducts was investigated using Taguchi’s L₁₆ (4⁵) experimental design. Protease type significantly (p < 0.05) influenced soluble yield, degree of hydrolysis (DH), angiotensin-I-converting enzyme (ACE) inhibitory activity and bitterness of hydrolysates, while percent enzyme only affected the DH. Hydrolysates produced by Alcalase and Protamex possessed strong ACE inhibitory activity (IC₅₀ = 100–200 μg/ml and 70 μg/ml, respectively), accompanied by high yield, high DH and strong bitterness. Furthermore, ACE inhibition was positively correlated (r² = 0.87) with bitterness of the hydrolysates. Fractionation by size-exclusion chromatography revealed that the bitter substances, which also showed strong ACE inhibition, were <3 kDa in size and contained many hydrophobic residues, including Tyr, Phe, Leu, Ile, Val and Lys. Despite the bitterness, these hydrolysates may have potential health benefits, arising from their potent ACE inhibitory activity.     

Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC–ESI-TOF MS/MS

Haruan myofibrillar protein was hydrolysed with proteinase K and thermolysin to isolate Angiotensin converting enzyme (ACE) inhibitory peptides. The thermolysin hydrolysate of myofibrillar protein with the highest ACE inhibition activity (IC₅₀ = 0.033 mg/ml) was fractionated by ultrafiltration and size exclusion chromatography to three fractions. Fraction F2 with higher ACE inhibitory activity was separated into five fractions (A–E) using reversed-phased high performance liquid chromatography (RP-HPLC). Fraction C showed 81% inhibition activity and was subjected to HPLC coupled to electrospray ionisation-time-of-flight mass spectrometry (ESI-TOF MS/MS). Two peptide sequences for the most abundant fragments were identified as VPAAPPK (IC₅₀ = 0.45 μM) at 791.155 m/z and NGTWFEPP (IC₅₀ = 0.63 μM) at 1085.841 m/z. The presence of two proline residues at the C-terminal sequence is responsible for the high ACE inhibitory activity of these peptides. The results suggest that Haruan meat protein hydrolysate is a potent ACE inhibitor and may be used to decrease blood pressure.     

Tyr-Pro-Lys,an angiotensin I-converting enzyme inhibitory peptide derived from broccoli (Brassica oleracea Italica)

To investigate the naturally occurring angiotensin I-converting enzyme (ACE) inhibitor, broccoli (Brassica oleracea Italica) extracts were used for its isolation and identification. After treatment with 50% acetone for membrane breakdown, ethyl acetate, n-butanol, and water were used for the preparation of broccoli extracts. The water-soluble extract from broccoli had 76.9% ACE inhibitory activity, while those of other organic solvent extracts showed lower ACE inhibitory activities. An ACE inhibitory peptide was isolated using column chromatographic methods including: Amberlite XAD-4, Sephadex LH-20, and high performance liquid chromatography. The purified ACE inhibitory peptide was identified to be a tripeptide, Tyr-Pro-Lys, having an IC₅₀ value of 10.5 μg protein/ml.