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.     

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 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.     

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).     

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 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.     

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.     

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.     

Amaranth seed protein hydrolysates have in vivo and in vitro antihypertensive activity

The objective of this work was to study the hydrolytic release of encrypted peptides with antihypertensive activity from storage proteins of Amaranthus mantegazzianus, as determined by in vitro assays, for the first time by in vivo studies in animal models, and by ex vivo assays. Hydrolysates with hydrolysis degree (DH) of 45% and 65% (IC50 0.12 mg/ml, equivalent to 300–600 μM) exhibited an angiotensin-I converting enzyme 1 (ACE) inhibitory activity equal or higher than the potential inhibitory of the average antihypertensive peptides registered in the BIOPEP database and of semi-purified Amaranthus hypochondriacus albumin and globulin protein fractions. Intragastric administration of hydrolysates with DH of 45% was effective in lowering blood pressure of male spontaneously hypertensive rats (SHR). Experiments performed in papillary muscles isolated from hearts and with isolated aortic smooth muscle of SHR suggest that the hypotensive effect could be attributed to a lowering of the peripheral resistance. We assume that the amaranth hydrolysates would be acting at the level of the local or autocrine renin–angiotensin system (RAS).     

Separation of angiotensin I‐converting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature,pH and digestive enzymes

Bovine collagen was isolated from connective tissue, a by‐product in the meat processing industry and characterised by SDS‐PAGE. Alcalase and papain were employed to generate collagen hydrolysates with different degree of hydrolysis (DH). In vitro angiotensin I‐converting enzyme (ACE) inhibitory activities were evaluated and the two most potent hydrolysates from each enzyme were separated by two‐step purification. Both alcalase‐catalysed and papain‐catalysed hydrolysates exhibited strong ACE inhibitory capacities with IC₅₀ values of 0.17 and 0.35 mg mL^?1, respectively. Purification by ion‐exchange chromatography and gel filtration chromatography revealed higher ACE inhibitory activities in one fraction from each enzyme with IC₅₀ values of 3.95 and 7.29 μg mL^?1. These peptide fractions were characterised as 6‐12 amino acid residues by MALDI‐TOF/MS. The peptides retained their activity (>90%) after exposure to processing temperature and pH and in vitro simulated gastrointestinal digestion. The present results demonstrated that collagen peptides can be utilised for developing high value‐added ingredients, for example ACE inhibitory peptides.     

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.     

Pilot-scale production of hydrolysates with altered bio-functionalities based on thermally-denatured whey protein isolate

Whey protein isolate (WPI) solutions (100 g L⁻¹ protein) were subjected to a heat-treatment of 80 °C for 10 min. Unheated and heat-treated WPI solutions were hydrolysed with Corolase^® PP at pilot-scale to either 5 or 10% degree of hydrolysis (DH). Hydrolysates were subsequently processed via cascade membrane fractionation using 0.14 μm, and 30, 10, 5 and 1 kDa cut-off membranes. The compositional and molecular mass distribution profiles of the substrate hydrolysates and membrane processed fractions were determined. Whole and fractionated hydrolysates were assayed for both angiotensin-I-converting enzyme (ACE) inhibitory activity and ferrous chelating capabilities. A strong positive correlation (P < 0.01) was established between the average molecular mass of the test samples and the concentration needed to chelate 50% of the iron (CC₅₀) in solution. The lowest ACE inhibition concentration (IC₅₀ = 0.23 g L⁻¹ protein) was determined for the 1 kDa permeate of the heat-treated 10% DH hydrolysate.     

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.     

Production of enzymatic hydrolysates with antioxidant and angiotensin-I converting enzyme inhibitory activity from pumpkin oil cake protein isolate

Protein isolate from pumpkin oil cake (PuOC PI) was hydrolysed by alcalase, flavourzyme and by sequential use of these enzymes, respectively, and the antioxidant properties and angiotensin-I converting enzyme (ACE) inhibitory activities of hydrolysates were evaluated. Under the same reaction conditions, alcalase hydrolysates showed a higher degree of hydrolysis (DH) than did flavourzyme hydrolysates. The highest DH’s by individual enzymes were 53.23 ± 0.7% and 37.17 ± 1.05%, respectively, both at 60 min. The increase of radical scavenging activity (RSA) in hydrolysates was positively correlated with the increase of DH, for both enzymes, though hydrolysates with flavourzyme showed two- or three-fold lower RSA than with alcalase. The highest bioactive potential was determined in the alcalase hydrolysate at 60 min, with RSA being 7.59 ± 0.081 mM TEAC/mg and ACE-inhibitory activity 71.05 ± 7.5% (IC₅₀ = 0.422 mg/ml). When this hydrolysate was further hydrolysed by flavourzyme, DH increased up to 69.29 ± 0.9%, but lower RSA (4.82 ± 0.21 mM TEAC/mg) and ACE-inhibitory activity (55.81 ± 6.196%) were determined in the final hydrolysate. This study suggested that the PuOC proteins could be converted into protein hydrolysates with antioxidant and ACE-inhibitory activities by enzymatic hydrolysis. Alcalase was shown as promising enzyme in further development of bioprocesses for the production of new bioactive food ingredients.     

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.     

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 angiotensin I-converting enzyme inhibitory peptides derived from soya milk

Inhibitors of angiotensin I-converting enzyme (ACE) are useful in treating hypertension, and many have been derived from food products, including soybeans. Using the industrial protease PROTIN SD-NY10, we developed a processed soya milk (PSM) with enhanced ACE inhibitory activity. The ACE inhibitory activity of PSM (IC₅₀ = 0.26 μg/ml) was greater than that of regular soya milk (IC₅₀ = 8.75 μg/ml). Eight novel ACE inhibitory peptides were purified from PSM by reversed-phase chromatography: FFYY (IC₅₀,1.9 μM), WHP (4.8 μM), FVP (10.1 μM), LHPGDAQR (10.3 μM), IAV (27.0 μM), VNP (32.5 μM), LEPP (100.1 μM), and WNPR (880.0 μM). The IC₅₀ values of these peptides are comparable to those reported for other ACE inhibitory peptides derived from wheat, chicken, bonito, wine, etc. Due to the relatively low IC₅₀ values of several peptides identified in this study, they may serve as ideal base components of food supplements for patients with hypertension.     

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 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.