Purification and characterization of angiotensin I-converting enzyme inhibitory peptides of small red bean (Phaseolus vulgaris) hydrolysates

Angiotensin I-converting enzyme (ACE) inhibitory activity was investigated for small red bean (Phaseolus vulgaris) protein hydrolysate produced by sequential digestion of Alcalase, papain followed by in vitro gastrointestinal simulation. The hydrolysate had ACE inhibitory activity with IC₅₀ of 67.2 ± 1.8 μg protein/mL. Peptides responsible for potent ACE inhibitory activity were isolated by a three-step purification process, including ultrafiltration, gel filtration and preparative reverse phase high performance chromatography (RP-HPLC). The fraction obtained after RP-HPLC fractionation with the highest activity yielded an IC₅₀ of 19.3 ± 1.4 μg protein/mL. Enzymatic kinetic studies using this fraction demonstrated competitive inhibition with K_i of 11.6 ± 1.7 μg protein/mL. Mass spectrometric characterization identified for the first time the octapeptide PVNNPQIH which demonstrated an IC₅₀ value of 206.7 ± 3.9 μM. The results expand the knowledge base of ACE inhibitory properties of small red bean protein hydrolysate and should be useful in further identification of specific ACE inhibitory peptides in beans.     

Structure–mechanism relationship of antioxidant and ACE I inhibitory peptides from wheat gluten hydrolysate fractionated by pH

The aims of this study were to assess bioactive properties (ACE inhibition and antioxidant capacity) from wheat gluten hydrolysate peptides fractionated by pH (4.0, 6.0 and 9.0), to determine peptide action mechanism, and to relate it to the secondary structure and functional groups of peptides. Gluten hydrolysate extracts (GHE) were enriched in peptides with medium hydrophobicity and molecular weight (≈ 60% MH and 5.5 kDa, respectively). Gluten peptides inhibited ACE I by uncompetitive mechanism and a direct relationship between α-helix structure and IC50% value was obtained (r = 0.9127). TEAC and cooper chelating activity from GHE 6.5 were the highest and directly correlated with MH peptides. GHE 9.0 had high carotene bleaching inhibition (47.5 ± 0.3%) and reducing power activity (163.1 ± 2.9 mg S₂O₃^2 − equivalent g^− 1 protein), which were directly related to disulfide bonds content of peptides (r = 0.9982 and 0.9216, respectively). pH was a good alternative to select bioactive peptides from wheat gluten hydrolysate.     

Isolation of antioxidative and ACE inhibitory peptides from protein hydrolysate of skipjack (Katsuwana pelamis) roe

Bioactive peptides from protein hydrolysate of defatted skipjack (Katsuwonus pelamis) roe with 5% degree of hydrolysis (DH) prepared by Alcalase digestion were isolated and characterised. Two active fractions with ABTS radical scavenging activity (973.01–1497.53 μmol TE/mg sample) and chelating activity (0.05–0.07 μmol EE/mg sample) from consecutive purification steps including ultrafiltration, cation exchange column chromatography and reverse phase high performance liquid chromatography (RP-HPLC), were subjected to analysis of amino acid sequence by LC–MS/MS. Seven dominant peptides with 6–11 amino acid residues were identified as DWMKGQ, MLVFAV, MCYPAST, FVSACSVAG, LADGVAAPA, YVNDAATLLPR and DLDLRKDLYAN. These peptides were synthesised and analysed for ACE-inhibitory activity and antioxidative activities. MLVFAV exhibited the highest ACE inhibitory activity (IC₅₀ = 3.07 μM) (p < 0.05) with no antioxidative property, whilst DLDLRKDLYAN showed the highest metal chelating activity, ABTS radical and singlet oxygen scavenging activities. Therefore, peptides prepared from skipjack roe could be further employed as a functional food ingredient.     

Angiotensin I-converting enzyme inhibitory activity of chickpea and pea protein hydrolysates

ACE inhibitory activity was studied for different hydrolysates obtained from protein concentrates of chickpea (kabuli and desi) and yellow pea (Golden) using in vitro gastrointestinal simulation, alcalase/flavourzyme, and papain. Protein/peptide profiles studied by SDS–PAGE and SE-HPLC, showed a rich composition of the hydrolysates in small peptides having MWs under 4 kDa. Papain hydrolysed yellow pea proteins showed the highest ACE inhibitory activity. In addition, chickpea desi proteins hydrolysed by in vitro gastrointestinal simulation showed higher ACE inhibition (IC₅₀ of 140 ± 1 μg/ml) compared to its digests obtained by alcalase/flavourzyme (IC₅₀ of 228 ± 3 μg/ml) or papain (IC₅₀ of 180 ± 1 μg/ml) and to chickpea kabuli hydrolysed by gastrointestinal simulation (IC₅₀ of 229 ± 1 μg/ml). The results demonstrate that enzymatic hydrolysates of chickpea and pea proteins contain bioactive ACE inhibitory peptides; furthermore, the type of enzyme used for hydrolysis affects the ACE inhibitory activity.     

Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation

Fresh low-fat milk was fermented with five mixed lactic acid bacteria for up to 30 h at 42 °C. A protease, prozyme 6, was added 5 h after the beginning of fermentation. The whey was separated from the fermented milk and freeze-dried. As the fermentation time extended to 30 h, soluble protein content increased from 30.9 to 195.9 mg g⁻¹, free amino acid content increased from 2.8 to 192.8 mg g⁻¹, peptide content increased from 6.4 to 402.8 mg g⁻¹ and γ-aminobutyric acid (GABA) increased from 0 to 80.6 mg 100 g⁻¹, while inhibition of angiotensin I-converting enzyme (ACE) increased as indicated by a decrease of IC₅₀ from 1.18 to 0.24 mg mL⁻¹, respectively. The amino acid sequences of two ACE inhibitory peptides were Gly–Thr–Trp and Gly–Val–Trp, of which the IC₅₀ values were 464.4 and 240.0 μm, respectively. The systolic blood pressure and diastolic blood pressure of spontaneously hypertensive rat (SHR) were reduced 22 and 21.5 mm Hg, respectively, after 8 weeks of oral administration of diluted whey (peptide concentration 5 mg mL⁻¹) from the 30 h fermentation.     

Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: Effect of substrate,enzyme and time of hydrolysis

Nine milk protein substrates were hydrolysed in vitro with five proteases for various times (0, 3, 6, and 24 h), and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates was assessed. Overall, the casein substrates gave rise to hydrolysates with significantly higher ACE-inhibitory activity than the whey protein (WP) substrates (85% vs. 79%). No significant difference between 3 and 24 h of hydrolysis was found. A reasonable correlation was found between the ACE inhibition of the 6 h hydrolysates determined in vitro and estimated by in silico modelling. The highest ACE-inhibitory activity was found in hydrolysates made with thermolysin followed by proteinase K, trypsin, pepsin and Bacillus licheniformis protease. The IC₅₀ values for thermolysin hydrolysates of caseins and WPs were 45–83 and 90–400 μg mL⁻¹, respectively, with α-lactalbumin giving the highest inhibitory activity. Thermolysin, proteinase K and trypsin were useful for the release of highly potent ACE-inhibitory peptides from both WPs and caseins.     

Production of novel ACE inhibitory peptides from β-lactoglobulin using Protease N Amano

Potent angiotensin I-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of β-lactoglobulin (βLg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to βLg f(36–42) and had an IC₅₀ value of 8 μm, which is considerably lower than the most potent ACE inhibitory peptides derived from bovine βLg reported so far.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from jellyfish Rhopilema esculentum

Two angiotensin I converting enzyme (ACE) inhibitory peptides were isolated from jellyfish Rhopilema esculentum protein hydrolysates prepared with pepsin and papain. Consecutive purification methods, including ion-exchange chromatography, size-exclusion chromatography and reverse-phase high-performance liquid chromatography, were used for isolation of ACE inhibitory peptides. The amino acid sequence of the peptides was identified as Gln-Pro-Gly-Pro-Thr and Gly-Asp-Ile-Gly-Tyr, respectively, and the IC₅₀ value of the purified peptides for ACE inhibitory activity was 80.67 μM and 32.56 μM. The purified peptides were evaluated for the antihypertensive effect in spontaneously hypertensive rats (SHR) after oral administration. Blood pressure decreased significantly after ingestion of the peptides. The results suggest that peptides derived from jellyfish R. esculentum may be beneficial as antihypertensive compounds in functional food resources.     

Tea and herbal infusions: Their antioxidant activity and phenolic profile

Tea and herbal infusions have been studied for their polyphenolic content, antioxidant activity and phenolic profile. The total phenolics recovered by ethyl acetate from the water extract, were determined by the Folin–Ciocalteu procedure and ranged from 88.1 ± 0.42 (Greek mountain tea) to 1216 ± 32.0 mg (Chinese green tea) GAE (Gallic acid equivalents)/cup. The antioxidant activity was evaluated by two methods, DPPH and chemiluminescence assays, using Trolox and quercetin as standards. The EC₅₀ of herbal extracts ranged from 0.151 ± 0.002 mg extract/mg DPPH (0.38 quercetin equivalents and 0.57 Trolox equivalents), for Chinese green tea, to 0.77 ± 0.012 mg extract/mg DPPH (0.08 quercetin equivalents and 0.13 Trolox equivalents), for Greek mountain tea. Chemiluminescence assay results showed that the IC₅₀ ranged from 0.17 ± 3.4 × 10⁻³ μg extract/ml of the final solution in the measuring cell (1.89 quercetin and 5.89 Trolox equivalents) for Chinese green tea, to 1.10 ± 1.86 × 10⁻² g extract/ml of the final solution in the measuring cell (0.29 quercetin and 0.90 Trolox equivalents) for Greek mountain tea. The phenolic profile in the herbal infusions was investigated by LC-DAD-MS in the positive electrospray ionization (ESI⁺) mode. About 60 different flavonoids, phenolic acids and their derivatives have been identified.     

Antihypertensive activity of milk fermented by Enterococcus faecalis strains isolated from raw milk

A total of 231 microorganisms were isolated from raw cow milk samples and the angiotensin-converting enzyme-inhibitory (ACEI) activity of the resultant fermented milk produced with the isolated microorganisms was assayed. Forty-six of these microorganisms were selected on the basis of high ACEI activity. Four Enterococcus faecalis strains stood out as producers of fermented milk with potent ACEI activity (IC₅₀ (the protein concentration that inhibits 50% of ACE activity): 34–59 μg mL⁻¹). Single doses (5 mL kg⁻¹) of the whey fraction obtained from these fermented milk samples were administered to spontaneously hypertensive rats (SHR) and to normotensive Wistar-Kyoto (WKY) rats in order to investigate their possible antihypertensive activity. Highly significant decreases in the systolic blood pressure (SBP) and in the diastolic blood pressure (DBP) were observed when the fermented milk was administered to SHR. Nevertheless, the fermented milk did not modify the SBP and the DBP of the WKY rats. Raw cow milk is an excellent source of wild lactic acid bacteria able to produce fermented milk with antihypertensive activity and antihypertensive activity of milk fermented by Enterococcus faecalis strains was associated with peptides different from Ile-Pro-Pro and Val-Pro-Pro.     

Three novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) using digestive proteases

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from muscle of cuttlefish (Sepia officinalis) by treatment with various digestive proteases were investigated. The most active hydrolysate was obtained with the crude protease extract from the hepatopancreas of cuttlefish (64.47 ± 1.0% at 2 mg of dry weight/ml) with a degree of hydrolysis of 8%. By gel filtration on Sephadex G-25 and RP-HPLC on C18 column, three novel peptides with high ACE-inhibitory activity were purified and their molecular masses and amino acid sequences were determined. The three peptides Val-Tyr-Ala-Pro, Val-Ile-Ile-Phe and Met-Ala-Trp with IC₅₀ values of 6.1, 8.7 and 16.32 μM, respectively, were novel ACE-inhibitory peptides. Lineweaver–Burk plots suggest that the three purified peptides act as non-competitive inhibitors against ACE. These results suggest that some peptides from cuttlefish could be a beneficial ingredient for nutraceuticals against hypertension.     

Isolation and characterisation of a novel antibacterial peptide from bovine αS1-casein

Bovine casein was hydrolysed with a range of proteolytic enzymes including pepsin, trypsin, α-chymotrypsin and β-chymotrypsin, and assessed for antibacterial activity. The pepsin digest of bovine casein, which showed antibacterial activity, was fractionated using reverse phase high performance liquid chromatography and the antibacterial peptides isolated were characterised using electrospray ionisation mass spectrometry. Two antibacterial peptides were identified, a novel peptide (Cp1) which corresponded to residues 99–109 of bovine α_S1-casein and a previously reported peptide (Cp2) which corresponded to residues 183–207 of bovine α_S2-casein. The minimum inhibitory concentration (MIC) of Cp1 and Cp2 were determined against a range of bacterial cultures. Cp1 exhibited an MIC of 125 μg mL⁻¹ against all Gram-positive bacteria tested, and MIC ranging between 125 and >1000 μg mL⁻¹ against the Gram-negative bacteria tested. Cp2 was generally far more potent against the Gram-positive bacteria, exhibiting an MIC of 21 μg mL⁻¹, compared to MICs ranging from 332 to >664 μg mL⁻¹ against most of the Gram-negative bacteria tested.     

Purification and hypotensive activity of rapeseed protein-derived renin and angiotensin converting enzyme inhibitory peptides

Rapeseed protein isolate (RPI) was hydrolyzed with Alcalase followed by reverse-phase high performance liquid chromatography (RP-HPLC) purification of bioactive peptides. The rapeseed protein hydrolysate (RPH) obtained after 4 h digestion with Alcalase had a degree of hydrolysis (DH) of ～11%. Gel permeation chromatography separation showed high contents of low molecular weight peptides in the RPH when compared to the RPI. After preparative and analytical RP-HPLC separations, three peptides (LY, TF and RALP) were purified and amino acid sequence determined by tandem mass spectrometry. LY (IC₅₀, 0.11 mM) was the most potent (p < 0.05) against ACE activity when compared to TF (IC₅₀, 0.81 mM) and RALP (IC₅₀, 0.65 mM). However, RALP (IC₅₀, 0.97 mM) was the most potent (p < 0.05) against renin activity when compared to LY (IC₅₀, 1.87 mM) and TF (IC₅₀, 3.1 mM). Single oral administration (30 mg/kg body weight) to spontaneously hypertensive rats showed LY and RALP to be the more effective hypotensive agents with maximum blood pressure reduction of ?26 and 16 mmHg, respectively when compared to TF (?12 mmHg). The results suggest that the higher number of hydrophobic amino acid residues LY and RALP contributed to their higher in vitro and in vivo activities when compared to TF.     

Studies on the anthocyanin profile and biological properties from the fruits of Acanthopanax senticosus (Siberian Ginseng)

The anthocyanin profile and biological activities, including antioxidant, xanthine oxidase inhibitory, angiotensin converting enzyme (ACE) inhibitory, and anticancer of Acanthopanax senticosus fruits were evaluated for the first time. The acidified 80% methanol extract of this species exhibited high biological properties at a concentration of 60 μg/ml. Moreover, cyanidin-3-O-(2″-O-xylosyl)-glucoside was identified using C₁₈ column chromatography, NMR spectroscopy, and HPLC-DAD-ESI/MS analysis. This compound was present at 5.2 mg/g, representing approximately 91% of the total peak area and possessed strong antioxidant effects against DPPH, ABTS, and hydroxyl radicals (IC₅₀ of 85.2, 43.7, and 126.6 μg/ml, respectively). Cyanidin-3-O-(2″-O-xylosyl)-glucoside also exhibited significant inhibitory activities against xanthine oxidase and ACE (IC₅₀: 55.5 and 47.1 μg/ml, respectively). Especially, LNCap (prostate), MOLT-4F (leukaemia), and ACHN (renal) cell lines exhibited potent anticancer effects, with IC₅₀ of 5.2, 11.2, and 22.5 μg/ml, respectively, in comparison with other cancer cell lines. Therefore, A. senticosus fruit may be utilised as an effective source for food and nutraceutical uses due to its high anthocyanin content as well as various biological properties.     

Partridgeberry polyphenols protect primary cortical and hippocampal neurons against β-amyloid toxicity

β-Amyloid (Aβ) deposition elicits a toxic effect on neurons and plays a crucial role in the etiology and/or progression of Alzheimer's disease (AD). Polyphenols found in fruits are endorsed for nutritional intervention in AD, since they are known to have extensive therapeutic properties apropos of brain health owing to their anti-oxidative effects against Aβ and neural reactive oxygen species (ROS). The present study was aimed to investigate the neuroprotective potential of polyphenols of partridgeberry (Vaccinium vitis-idaea L.) and elucidate the mechanism by which they confer protection against Aβ toxicity in rat primary neurons in vitro. The pre-treatment of rat primary cortical and hippocampal neurons with partridgeberry polyphenols (10–200 μg mL^− 1) significantly attenuated Aβ-induced cell death and membrane damage. The flavan-3-ol- and flavonol-rich fractions of the partridgeberry exhibited the strongest ability to maintain cell viability (EC₅₀ 5.9 μg mL^− 1) and prevent lactose dehydrogenase release (IC₅₀ 0.01 μg mL^− 1) (P ≤ 0.05). Similar to the maintenance of cellular viability, the flavan-3-ol- and flavonol-rich fractions also amplified the greatest activity of SOD and catalase among all polyphenol preparations exposed to neurons (P ≤ 0.05). All four partridgeberry polyphenol preparations reduced the intracellular Aβ levels by 7–15 folds, and initiated Aβ clearance from neurons as compared to untreated cells (P ≤ 0.05). Partridgeberry derived polyphenol preparations; especially the flavonol-rich fraction (IC₅₀ 97.1 μg mL^− 1) significantly modulated the apoptotic targets and in vitro acetylcholinesterase activity (P ≤ 0.05), indicating potential pharmacotherapy application in AD. Furthermore, the restoration of hyperactive caspases and Bcl2 family of apoptotic architects added to the neuroprotective candidacy of PPFs. These findings suggest that partridgeberry polyphenols, especially flavan-3-ol- and flavonol-rich fractions, could be of importance in prevention and/or treatment of AD.     

Determination of lactoferrin in bovine milk,colostrum and infant formulas by optical biosensor analysis

An automated, rapid, sensitive and label-free biosensor-based immunoassay for lactoferrin in bovine milk utilising surface plasmon resonance (SPR) optical detection is described. Lactoferrin content is estimated from its specific interaction with an anti-bovine lactoferrin antibody immobilised on the sensor surface in a direct- binding assay format. Samples are prepared for analysis by direct dilution into buffer. Analysis conditions, including ligand immobilisation, flow-rate, contact time and regeneration have been defined and non-specific binding considerations evaluated. Performance parameters include a working range of 0–1000 ng mL⁻¹, a method-detection limit of 19.9 μg mL⁻¹ in undiluted milk, overall instrument response RSD_R of 3.50%, a mean inter-assay RSD_R of 10.8% for milk and surface stability over ca 500 samples. The technique was applied to the measurement of lactoferrin content of consumer milks, colostrum and infant formulas, and temporal change during early bovine lactation.     

Available lysine,protein digestibility and lactulose in commercial infant formulas

Available lysine, in vitro protein digestibility and lactulose values were determined in 23 commercial infant formulas. The mean available lysine content of the formulas based on dairy proteins was 66.7±9.5 mg g⁻¹ protein, similar to that of human milk, while that of soy based formulas was considerably lower (45.0±8.3 mg g⁻¹ protein). In vitro protein digestibility values ranged 85.5–88.9% for soy-based formulas and 90.5–98.3% for formulas based on dairy proteins. Formulas based on milk enriched with whey had higher lactulose content than those based on cow's milk. However, all values were below the limit of 600 mg L⁻¹ recommended for UHT milk.     

Optimization of sour milk fermentation for the production of ACE-inhibitory peptides and purification of a novel peptide from whey protein hydrolysate

The effect of fermentation conditions on the production of angiotensin-I converting enzyme (ACE) inhibitory peptide in sour milk fermented by Lactobacillus helveticus LB10 was investigated using response-surface methodology. Optimal conditions to produce the maximum production of ACE-inhibitory peptides were found to be 4% (v/w) inoculum, 7.5 initial pH of medium and 39.0 °C. The fermented milk resulted in 75.46% inhibition in ACE activity. The cell-envelope proteinase, assisted by X-prolyldipeptidyl aminopeptidase of Lb. helveticus LB10 produced the ACE-inhibitory peptides. A novel ACE-inhibitory peptide from whey protein hydrolysate produced by crude proteinases of Lb. helveticus LB10 was purified. The separations were performed with Sephadex^® G-75 and Sephadex G-15 gel filtration chromatography and reversed-phase, high-performance liquid chromatography. The peptide with the RLSFNP sequence was isolated from β-lactoglobulin hydrolysate and its IC₅₀ while inhibiting ACE activity was 177.39 μm.     

Release of multifunctional peptides by gastrointestinal digestion of sea cucumber (Isostichopus badionotus)

Sea cucumber is a benthic marine organism distributed worldwide and used as food in several Asian countries. The species Isostichopus badionotus is captured intensively off the Yucatan Peninsula, Mexico. Boiled I. badionotus was subjected to in vitro simulated gastrointestinal digestion using pepsin and a pepsin–Corolase PP^® mixture. ACE-inhibitory and radical scavenging activities, iron reducing capacity and cytotoxic effects against colorectal cancer cells were evaluated in the hydrolysates and their ultrafiltered fractions. ACE-inhibitory activity was potent in fractions containing peptides <3000 Da, an effect augmented with combined action of gastric (pepsin) and intestinal (Corolase PP^®) enzymes (IC₅₀ = 0.038 ± 0.004 mg/mL). Antioxidant activity was exerted by peptides with low and high molecular weights, depending on hydrolysis method. This is the first report of cytotoxic capacity against colorectal HT-29 cells in peptides from sea cucumber. Sea cucumber hydrolysates and ultrafiltered fractions are potential ingredients for development of functional foods.     

Inhibitory effect of polyphenol-rich extracts of jute leaf (Corchorus olitorius) on key enzyme linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin I converting) in vitro

Corchorus olitorius leaf is consumed in various parts of the world as leafy vegetable and folk remedy for the management of some degenerative diseases with dearth of information on its biochemical rationale. Therefore, this study sought to characterize the inhibitory action of polyphenol-rich extracts (free and bound) of C. olitorius on α-amylase, α-glucosidase and angiotensin I converting enzyme (ACE), as well as to identify the phenolic compound responsible for these activities. Our findings revealed that the extracts inhibited α-amylase and α-glucosidase (12.5–50.0 μg/mL), and ACE (10.0–50.0 μg/mL) in dose-dependently with free extracts having significantly (P < 0.05) higher α-amylase (17.5 μg/mL), α-glucosidase (11.4 μg/mL) and ACE (15.7 μg/mL) inhibitory activities as revealed by the IC₅₀. Reversed-phase HPLC analysis of the extracts revealed chlorogenic acid (7.5 mg/100 g) and isorhamnetin (51.1 mg/100 g) as the main phenolics in the free extract and caffeic acid (58.1 mg/100 g) in the bound extract. Therefore, the enzyme inhibitory activity of C. olitorius extracts may be attributed to the presence of caffeic acid, chlorogenic acid and isorhamnetin, thus justifying its use in folklore for the management of diabetes and hypertension.