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.     

Angiotensin I-converting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats

In order to utilize yellowfin sole (Limanda aspera) frame protein, which is normally discarded as industrial waste in the process of fish manufacture, yellowfin sole frame protein was hydrolysed by α-chymotrypsin. Yellowfin sole frame protein hydrolysates (YFPHs) were fractionated into three ranges of molecular weight (YFPH-I, 30–10 kDa; YFPH-II, 10–5 kDa; YFPH-III, below 5 kDa) using an ultrafiltration (UF) membrane bioreactor system. Angiotensin I-converting enzyme (ACE) inhibitory activity was detected on YFPH-III, and the ACE inhibitory peptide (YFP) was purified from YFPH-III using consecutive chromatographic techniques. The YFP with a molecular mass of 1.3 kDa consisted of 11 amino acids, Met-Ile-Phe-Pro-Gly-Ala-Gly-Gly-Pro-Glu-Leu, and its IC₅₀ value was 28.7 μg/ml. Lineweaver–Burk plots suggest that YFP acts as a non-competitive inhibitor to inhibit ACE. Antihypertensive effects of YFP on spontaneously hypertensive rats (SHR) following oral administration was determined as the blood pressure significantly decreased after peptide ingestion.     

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.     

Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate

Our objective was to evaluate the angiotensin I converting enzyme (ACE) inhibitory activity of skate skin protein hydrolysates and its corresponding fractions. The skate skin hydrolysates were obtained by enzymatic hydrolysis using alcalase, α-chymotrypsin, neutrase, pepsin, papain, and trypsin. Amongst the six hydrolysates, the α-chymotrypsin hydrolysate had the highest ACE inhibitory activity compared to other hydrolysates. The amino acid sequences of the purified peptides were identified to be Pro–Gly–Pro–Leu–Gly–Leu–Thr–Gly–Pro (975.38 Da), and Gln–Leu–Gly–Phe–Leu–Gly–Pro–Arg (874.45 Da). The purified peptides from skate skin had an IC₅₀ value of 95 μM and 148 μM, respectively, and the Lineweaver–Burk plots suggest that they act as a non-competitive inhibitor against ACE. Our study suggested that novel ACE inhibitory peptides derived from skate skin protein may be beneficial as anti-hypertension compounds in functional foods.     

Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides

Yak (Bos grunniens) milk casein derived from Qula, a kind of acid curd cheese from northwestern China, was hydrolysed with alcalase. The hydrolysates collected at different hydrolysis times (0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min) were assayed for the inhibitory activity of angiotensin-I-converting enzyme (ACE), and the one obtained at 240 min hydrolysis showed the highest ACE inhibitory activity. The active hydrolysate was further consecutively separated by ultrafiltration with 10 kDa and then with 6 kDa molecular weight cut-off membranes into different parts, and the 6 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Pro–Pro–Glu–Ile–Asn (PPEIN)(κ-CN; f156–160) and Pro–Leu–Pro–Leu–Leu (PLPLL) (β-CN; f136–140), respectively. The molecular weight and IC₅₀ value of the peptides were 550 Da and 566.4 Da, and 0.29 ± 0.01 mg/ml and 0.25 ± 0.01 mg/ml, respectively.     

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.     

Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase^®, chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2 ± 1.5% at 2 mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Biological functions of all fractions were assayed, and P₄ was found to display a high ACE inhibitory activity. The IC₅₀ values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P₄ were 1.2 ± 0.09 and 0.81 ± 0.013 mg/ml, respectively. Further, P₄ showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P₄ was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine.     

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.     

Isolation and characterisation of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from the algae protein waste

A hendeca-peptide with angiotensin I-converting enzyme (ACE) inhibitory activity was isolated from the pepsin hydrolysate of algae protein waste, a mass-produced industrial by-product of an algae essence from microalgae, Chlorella vulgaris. Edman degradation revealed its amino acid sequence to be Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe. Inhibitory kinetics revealed a non-competitive binding mode with IC₅₀ value against ACE of 29.6 μM, suggesting a potent amount of ACE inhibitory activity compared with other peptides from the microalgae protein hydrolysates which have a reported range between 11.4 and 315.3 μM. In addition, the purified hendeca-peptide completely retained its ACE inhibitory activity at a pH range of 2–10, temperatures of 40–100 °C, as well as after treatments in vitro by a gastrointestinal enzyme, thus indicating its heat- and pH-stability. The combination of the biochemical properties of this isolated hendeca-peptide and a cheap algae protein resource make an attractive alternative for producing a high value product for blood pressure regulation as well as water and fluid balance.     

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.     

ACE-inhibitory activity of tilapia protein hydrolysates

Fish processing wastes can be used for preparing bioactive peptides with various functionalities. Our objective was to evaluate the in vitro angiotensin converting enzyme (ACE) inhibitory activity of tilapia protein hydrolysates and its corresponding fractionates. Tilapia protein was alkali-solubilised at pH 11.0 and recovered at pH 5.5 to obtain a stable substrate. This substrate was hydrolysed using two enzymes, Cryotin-F or Flavourzyme, to 7.5% and 25% degree of hydrolysis (DH). The hydrolysates were ultra-filtered into three fractions: >30 kDa fraction, 10–30 kDa fraction, and <10 kDa fractions. Both hydrolysates and fractionates were tested for ACE inhibition. Results showed that both Cryotin and Flavourzyme hydrolysates with 25% DH gave maximum ACE inhibitory activity. Low MW peptides showed higher ACE inhibition than high MW peptides. The inhibitory activity of fractionates was lower than that of the corresponding hydrolysates, possibly due to separation and removal of synergistic peptides by ultrafiltration. Amongst fractionates, all the 7.5% DH Cryotin fractions and 25% DH Flavourzyme fractions exhibited optimum % ACE inhibition. The results of this research could be used for optimising enzyme parameters to obtain peptides from tilapia with optimum in vitro ACE inhibitory activity.     

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).     

Chemical composition,angiotensin I-converting enzyme (ACE) inhibitory,antioxidant and antimicrobial activities of the essential oil from Periploca laevigata root barks

The present study describes the chemical composition, and antimicrobial, antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of essential oil from Periploca laevigata root barks (PLRB), an aromatic plant widely distributed in Tunisia and used as a traditional medicinal plant. Gas chromatography/mass spectrometry was used to determine the composition of the PLRB oil. Forty-three components were identified in the essential oil and the main compounds were benzaldehyde (56%), methyl 4-methoxysalicylate (6.55%) and carvacrol (4.75%). The PLRB essential oil exhibited a dose-dependent manner of inhibitory activity toward ACE. The highest ACE inhibitory activity (54%) was observed at a concentration of 30 μg/ml. The PLRB oil was also found to possess antioxidant activities, as evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, β-carotene bleaching and reducing power assays. The antimicrobial activity of the essential oil was also investigated on several microorganisms. The inhibition zones and minimal inhibitory concentration (MIC) values of bacterial strains were in the range of 12–46 mm and 50–300 μg/ml, respectively. The inhibitory activity of the PLRB essential oil against Gram-positive bacteria was significantly higher than against Gram-negative. It also exhibited remarkable activity against several fungal strains.     

Antioxidant properties of peptide fractions from silver carp (Hypophthalmichthys molitrix) processing by-product protein hydrolysates evaluated by electron spin resonance spectrometry

Silver carp processing by-product protein is usually discarded as an industrial solid waste. In this study the protein was recovered using a pH-shift method, after which seven commercial proteases were separately employed to prepare antioxidative hydrolysates. Among the hydrolysates, pepsin hydrolysates, which had the highest free radical-scavenging activity, were further separated into five peptide fractions, SCPH-I (>10 kDa), SCPH-II (5–10 kDa), SCPH-III (3–5 kDa), SCPH-IV (1–3 kDa), and SCPH-V (<1 kDa), by using ultrafiltration. The antioxidative properties of the peptide fractions were investigated, using a free radical-scavenging assay, by electron spin resonance. The results show that SCPH-V had the highest scavenging effects on DPPH (1,1-diphenyl-2-picrylhydrazyl), hydroxyl and superoxide anion radicals. SCPH-V had potent antioxidant activity in the prevention of the peroxidation of linoleic acid and alleviation of H₂O₂-induced oxidative stress in human intestinal epithelial caco-2 cells. The results indicated that the antioxidant capacity of silver carp by-product hydrolysates could be enhanced by ultrafiltration.     

Identification of pro-drug type ACE inhibitory peptide sourced from porcine myosin B: Evaluation of its antihypertensive effects in vivo

This study aimed to identify a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from porcine skeletal myosin B. Proteins were hydrolyzed with pepsin and the hydrolysates were then subjected to various types of chromatography to isolate the active peptides. The 50% inhibitory concentrations of Lys–Arg–Val–Ile–Gln–Try (M6 a novel peptide) and Val–Lys–Ala–Gly–Phe (A5, a peptide discovered by Ukeda et al. (1991)) were 6.1 and 20.3 μM, respectively. As a result of a homology search, it was determined that the M6 peptide originated from myosin and peptide A5 was of actin origin. M6 is a novel ACE inhibitory peptide, whose activity was shown to be the strongest amongst the previously published myosin-originated peptides. Kinetic evaluations showed that both peptides are competitive inhibitors of ACE. Based on their activity against ACE, M6 was classified as a pro-drug conformer and A5 was classified as a substrate conformer. When both peptides were administered orally to spontaneously hypertensive rats at doses of 10 mg/kg, temporal hypertension was observed after 6 h. This study suggests that M6 and A5 are peptides that may serve several purposes. Based on their remarkable antihypertensive activity, we suggest that M6 and A5 may have potential applications as functional food, which could be used as nutraceutical compounds.     

α-Glucosidase inhibitors from Chinese Yam (Dioscoreaopposita Thunb.)

The inhibitory activities of crude extracts and purified constituents from the fresh tuberous rhizomes of Chinese Yam (Dioscorea opposita Thunb.), which is commonly called Huai Shan Yao in Chinese, were evaluated against yeast α-glucosidase in order to search for the active principals for treatment of diabetes. Bioassay-guiding isolation gave four compounds: trans-N-p-coumaroyltyramine (1) (IC₅₀ = 0.40 μM), 1,7-bis(4-hydroxyphenyl)heptane-3,5-diol (2) (IC₅₀ = 0.38 mM), 6-hydroxy-2,4,7-trimethoxyphenanthrene (3) (IC₅₀ = 0.77 mM) as α-glucosidase inhibitors, and cis-N-p-coumaroyltyramine (4), an isomer of compound 1, which showed no inhibitory activity against α-glucosidase. Furthermore, the separation and purification of compound 3 from Chinese Yam (Huai Shan Yao) was conducted by high-speed counter-current chromatography (HSCCC) using hexane–ethyl acetate–methanol–water (1:1:1:1, v/v/v/v). Compound 1, 2 and 4 were isolated from or detected in the Dioscoreaceae family for the first time.     

Antihypertensive effect of a bovine lactoferrin pepsin hydrolysate: Identification of novel active peptides

The potential of bovine lactoferrin (LF) as a source of antihypertensive peptides has been examined. For this purpose, LF pepsin hydrolysate with molecular mass lower than 3 kDa (LFH < 3 kDa) was prepared and orally administered to spontaneously hypertensive rats (SHR), resulting in reduced systolic blood pressure in a significant and maintained manner up to 24 h after administration. LFH < 3 kDa was further fractionated by semi-preparative high performance liquid chromatography (HPLC) and 38 peptides, contained in the active fractions, were identified by using an ion trap mass spectrometer. Based on the peptide abundance, a total of 11 peptides were chemically synthesized and their ACE inhibitory activity tested. Only three of them, corresponding to peptides of sequences LIWKL, RPYL and LNNSRAP exerted in vitro inhibitory effects on angiotensin I converting enzyme (ACE) activity and had a 50% inhibitory concentration (IC₅₀) of 0.47, 56.5 and 105.3 μM, respectively. The three peptides also showed antihypertensive effects in SHR and remarkably the effect of LIWKL remained significant for up to 24 h post-administration, similarly LFH < 3 kDa and the captopril control. The two most potent in vitro inhibitory peptides showed ex vivo inhibitory effect on ACE-dependent vasoconstriction as well. In conclusion, three novel LF-derived peptides and a pepsin LFH < 3 kDa lowered blood pressure and exhibit potential as orally effective antihypertensive compounds.     

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.     

Influence of degree of hydrolysis on functional properties,antioxidant activity and ACE inhibitory activity of peanut protein hydrolysate

Functional properties, antioxidant and Angiotensin-converting enzyme (ACE) inhibitory activities of peanut protein isolate (PPI) and peanut protein hydrolysate (PPH) prepared using Alcalase, at different (10%, 20%, 30% and 40%) degrees of hydrolysis, (DH) were investigated. Hydrolysis (at DH > 10%) significantly (p < 0.05) improved the solubility (>80%) of PPI, especially in the pH range of 4–6. However, PPI showed better emulsifying and foaming properties than PPH (p < 0.05). As DH increased, ferrous ion chelating activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and ACE inhibitory activity of PPH increased, while reducing power decreased (p < 0.05). Bleaching of beta-carotene by linoleic acid was suppressed better by PPI and PPH at 10% DH than of PPH at higher DH. Thus, the results reveal that DH affects functional properties, antioxidant and ACE inhibitory activities of peanut protein.