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.     

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.     

Influence of temperature and degree of hydrolysis on the peptide composition of trypsin hydrolysates of β-lactoglobulin: Analysis by LC–ESI-TOF/MS

Enzymatic hydrolysis of proteins is influenced, either positively or negatively, by the hydrolysis conditions, temperature, enzyme concentration and pH, as well as substrate pre-treatments, e.g. heat-denaturing, glyco-conjugation and/or cross-linking. Purified bovine β-lactoglobulin (96.0% nitrogen) was hydrolysed using trypsin (EC 3.4.21.4, bovine pancrease) at between 30 and 50 °C to degrees of hydrolysis (DHs) between 1 and ∼9.0%. The time taken to reach the desired DH varied greatly, being shortest at 45 and 50 °C and longest at 30 °C. The hydrolysates were analysed by tandem liquid chromatography–electrospray ionisation time-of-flight mass spectra (LC–ESI-TOF/MS) and results showed that the detectable peptides, at both 30 °C and 35 °C, were similar at DH 1%. However, not only were the detectable peptides produced at 40–50 °C different from those produced at lower temperatures, but the trypsin released peptides due to non-specific hydrolysis of β-Lg. The pattern resembled a shift of trypsinolysis towards chymotrypsinolysis, probably due to steric ‘stretching’ and increase of the catalytic pocket, thus allowing bulky amino acids to be processed. Hydrolysis at 30 °C to DH 5% and 10% also led to the release of peptides due to non-specific cleavage by trypsin. These results indicate that trypsin could only release peptides in a predictable manner at temperatures near, but lower than, the declared optimum of 37 °C. Above this temperature and above DH 5–10% at 30 °C, hydrolysis followed a mixed trypsin- and chymotrypsin-like activity. Lys–Pro, Lys–Ile(–Pro) and Lys–Phe bonds remained stable to trypsin at all temperatures. Some peptides with a high content of hydrophobic amino acids were undetected by ESI-TOF/MS, probably due to their poor ionisation.     

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.     

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 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.     

Production,in continuous enzymatic membrane reactor,of an anti-hypertensive hydrolysate from an industrial alfalfa white protein concentrate exhibiting ACE inhibitory and opioid activities

A few hydrolysates of food proteins were reported to exert an antihypertensive effect in vivo due to angiotensin I converting enzyme (ACE) inhibitory peptides or opioid peptides. An industrial alfalfa white protein concentrate (AWPC) hydrolysate, produced on a pilot plant scale, strongly inhibited ACE with an IC50 value of 8.8 μg/ml. Furthermore, the AWPC hydrolysate inhibited the spontaneous contraction of rabbit ileum in a dose-dependent and naloxone-blocked manner, indicating the presence of opioid peptides. The AWPC hydrolysate significantly lowered blood pressure of spontaneously hypertensive rats (SHR) more than 6 h after a single oral administration. The maximum reduction of systolic blood pressure (−29.9 ± 2.0 mmHg) was observed 4 h after an oral administration of 0.5 g/kg hydrolysate dosage. The minimal anti-hypertensive effect was observed for 0.1 g/kg dosage. The AWPC hydrolysate may then be particularly suitable as nutraceutical for functional food useful in the prevention of hypertension.     

Some functional properties of oat bran protein concentrate modified by trypsin

Oat bran protein concentrate (OBPC) was prepared from oat bran, and hydrolyzed using trypsin. Protein hydrolysates of three different degrees of hydrolysis (4.1%, 6.4% and 8.3% respectively) were obtained. SDS-PAGE analysis demonstrated that oat globulin was the major protein component in OBPC. After trypsin treatment, acidic polypeptides were partly degraded into large peptides (M_r = 29,000–33,000) and small peptides (M_r < 20,000); however, basic polypeptides were almost intact. The functional properties of the resulting products were compared with those of control OBPC. Marked changes in the protein functionality were caused by proteolysis. The solubility, water-holding capacity, emulsifying activity and foaming ability of the hydrolysates gradually increased with the increase in DH. However, the oil-holding capacity, emulsifying stability and foaming stability of the hydrolysates reduced to a certain extent.     

Antihypertensive activity of AMC3, an engineered 11S amaranth globulin expressed in Escherichia coli,in spontaneously hypertensive rats

The acidic-subunit of 11S seed storage globulin of Amaranthus hypochondriacus was recently modified by the incorporation of antihypertensive biopeptides: four units of Val–Tyr dipeptides (VY) in tandem and one of Ile–Pro–Pro tripeptide (IPP), was named AMC3. The in vivo effect of AMC3 enzymatic hydrolysates (AEH) produced in Escherichia coli, was evaluated in spontaneously hypertensive rats (SHR) by once-oral administration experiments. As positive control a SHR group received a dose of captopril (50 mg/kg) (an angiotensin-converting enzyme (ACE) inhibitor used for the treatment of hypertension). Mean arterial pressure (MAP) was measured for 6 h after AEH or captopril administration. AEH administration at maximal dose (100 mg/kg) significantly reduced MAP similar to the group treated with captopril. The maximal reduction in MAP was observed after 3.5 h after AEH oral administration. The present study showed that enzymatic hydrolysates of AMC3 containing ACE inhibitory peptides (4xVY and IPP) sequences had significant antihypertensive action by oral administration in spontaneously hypertensive rats (SHR).     

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.     

Fatty acid synthase and in vitro adipogenic response of human adipocytes inhibited by α and α′ subunits of soybean β-conglycinin hydrolysates

The objective was to assess the effect of protein hydrolysates of β-conglycinin enriched soybean on fatty acid synthase (FAS) activity and adipogenic response of human adipocytes in vitro. The results showed that genotypic changes in soybean protein subunits produced peptide profiles that led to inhibition of FAS and lipid accumulation in vitro. FAS inhibitory potency (IC₅₀) of soy protein hydrolysates (SPH) ranged from 50 to 175 μM, while lipid inhibition from 15.6% to 45.9%. Protein hydrolysate C2H from a soybean containing the highest total β-conglycinin (46.9%) showed the most potent inhibitory effect on in vitro adipogenesis (46%) and FAS (IC₅₀ = 50 μM). C2H was composed of dominant peptides from fragments f(85–112) and f(131–132) of β-conglycinin α subunit. Smaller peptides identified as fragments f(330–342) and f(329–342) of α′ subunit were also found. In conclusion, soybean genotypes enriched in β-conglycinin α and α′ subunits are suitable sources of active peptides that inhibit FAS activity and lipid accumulation.     

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.     

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.     

Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process

The overall aim of this work was to characterise the major angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilisation of β-lactoglobulin and casein-derived peptides (CDP) from whey. The adsorbed proteins were hydrolysed in situ, which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC₅₀ = 287 μg/mL) and (ii) from β-lactoglobulin (IC₅₀ = 128 μg/mL). The well-known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterised in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the bitterness of the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC₅₀).     

Antioxidative activity and functional properties of protein hydrolysate of yellow stripe trevally (Selaroides leptolepis) as influenced by the degree of hydrolysis and enzyme type

Antioxidative activity and functional properties of protein hydrolysates from yellow stripe trevally (Selaroides leptolepis) meat, hydrolyzed by Alcalase 2.4L (HA) and Flavourzyme 500L (HF) with different degrees of hydrolysis (DH) were investigated. As the DH increased, DPPH radical-scavenging activity and reducing power of HA decreased (p < 0.05) but no differences were observed for HF (p > 0.05). Metal chelating activity of both HA and HF increased with increasing DH (p < 0.05). HF generally had a higher (p < 0.05) chelating activity than had HA at the same DH tested. At low DH (5%), HA exhibited a better DPPH radical-scavenging activity while, at high DH (25%), HF had a higher (p < 0.05) reducing power. For the functional properties, hydrolysis by both enzymes increased protein solubility to above 85% over a wide pH range (2–12). When the DH increased, the interfacial activities (emulsion activity index, emulsion stability index, foaming capacity, foam stability) of hydrolysates decreased (p < 0.05), possibly caused by the shorter peptide chain length. At the same DH, the functionalities of protein hydrolysate depended on the enzyme used. The results reveal that antioxidative activity and functionalities of protein hydrolysates from yellow stripe trevally meat were determined by the DH and by the enzyme type employed.     

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.     

Anticoagulant activities of goby muscle protein hydrolysates

The anticoagulant activities of protein hydrolysates prepared from goby muscle by treatment with various bacterial alkaline proteases were investigated. All proteases exhibited varying degrees of hydrolysis (DH) and all goby protein hydrolysates (GPHs) caused a significant prolongation of both the thrombin time (TT) and the activated partial thromboplastin time (APTT). The hydrolysate generated by the crude protease from Bacillus licheniformis NH1 displayed the highest anticoagulant activity, and the higher TT (about 32 s) at a concentration of 5 mg/mL was obtained with hydrolysate having a DH of 8.86%. This hydrolysate was then fractionated by size exclusion chromatography on a Sephadex G-25 column into five major fractions (F1–F5). Fraction F2, which exhibited the highest anticoagulant activity, was then fractionated by reversed-phase high-performance liquid chromatography. The molecular masses and amino acid sequences of four peptides in peptide sub-fraction F2–6, which exhibited the highest anticoagulant activity, were determined using ESI-MS and ESI-MS/MS, respectively. The structures of these peptides were identified as Leu-Cys-Arg, His-Cys-Phe, Cys-Leu-Cys-Arg and Leu-Cys-Arg-Arg.     

Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe

Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe, hydrolysed by Alcalase 2.4 L (RPH) with different degrees of hydrolysis (DH) at various concentrations were examined. As DH increased, the reduction of DPPH, ABTS radicals scavenging activities and reducing power were noticeable (p < 0.05). The increases in metal chelating activity and superoxide scavenging activity were attained with increasing DH (p < 0.05). However, chelating activity gradually decreased at DH above 30%. All activities except superoxide anion radical scavenging activity increased as the concentration of hydrolysate increased (p < 0.05). Hydrolysis using Alcalase could increase protein solubility to above 80% over a wide pH range (2–10). The highest emulsion ability index (EAI) and foam stability (FS) of hydrolysates were observed at low DH (5%) (p < 0.05). Concentrations of hydrolysates determined interfacial properties differently, depending on DH. The molecular weight distribution of RPH with 5%DH (RPH5) was determined using Sephadex G-75 column. Two major peaks with the molecular weight of 57.8 and 5.5 kDa were obtained. Fraction with MW of 5.5 had the strongest metal chelating activity and ABTS radical scavenging activity. The results reveal that protein hydrolysates from defatted skipjack roe could be used as food additives possessing both antioxidant activity and functional properties.     

Novel peptides derived from egg white protein inhibiting alpha-glucosidase

The aim of this study was to search and identify potential anti-diabetic peptides with α-glucosidase and α-amylase inhibitory activities. After the Alcalase hydrolysis, egg white protein hydrolysates were purified and identified by LC–MS–MS. Eight identified peptides were further synthesized by the Fmoc solid-phase synthesis. The anti-diabetic activities of these synthetic peptides were assessed using enzymatic inhibitory assays against the α-glucosidase and α-amylase. Among the eight peptides, peptide RVPSLM was discovered as a potential α-glucosidase inhibitor with an IC₅₀ value at 23.07 μmol L⁻¹. However, it did not exhibit a visible or detectable inhibitory efficiency on the α-amylase. These studies indicate the potential of using egg white protein hydrolysates as a functional food product with the anti-diabetic activity.     

The use of high hydrostatic pressure to promote the proteolysis and release of bioactive peptides from ovalbumin

Enzymatic hydrolysis of food proteins can release peptides able to exert different biological activities. Among the bioactive peptides known so far, those with angiotensin converting enzyme (ACE)-inhibitory properties are receiving special attention due to their potential beneficial effects in the treatment of hypertension. In a previous work, we identified active peptide sequences that derived from proteolysis of ovalbumin. We have now explored the possibility of using high hydrostatic pressure to promote the release of bioactive peptides. Treatment of ovalbumin under high pressures, up to 400 MPa, with chymotrypsin, trypsin and pepsin, enhanced its hydrolysis and changed the proteolytic pattern. However, under the conditions assayed, the in vitro ACE inhibitory activity of the hydrolysates did not improve as compared with those obtained at atmospheric pressure. Nevertheless, proteolysis under pressures of 200–400 MPa accelerated the release of the peptides YAEERYPIL, FRADHPFL and RADHPFL, with demonstrated antihypertensive effects in vivo.