Angiotensin I‐converting enzyme inhibitory and Ca‐binding activities of peptides prepared from tuna cooking juice and spleen proteases

Tuna cooking juice is a by‐product from the tuna canning industry. In this study, tuna cooking juice was hydrolysed by proteases extracted from the spleen. Tuna cooking juice showed the highest ACE inhibitory and Ca‐binding activities after hydrolysis for 270 and 180 min, respectively. The hydrolysate was further fractionated by ultrafiltration. The permeate exhibited highest ACE inhibitory and Ca‐binding activities when passed through 1 and 5 kDa cut‐off membranes, respectively. Gel filtration chromatography was used to determine the MW of bioactive peptides that exhibited highest ACE inhibitory and Ca‐binding activities. Those peptides that exhibited highest ACE inhibitory and Ca‐binding activities were the MW range of 238–829 Da and 1355–1880 Da, respectively. These results suggest that the tuna cooking juice and the spleen protease extract are a potential source of bioactive peptides that can be utilised as bioactive ingredients in functional food and nutraceuticals.     

Isolation and characterization of angiotensin I‐converting enzyme inhibitory peptides from wheat gliadin hydrolysate

Angiotensin I‐converting enzyme (ACE) inhibitory peptide was isolated from wheat gliadin hydrolysate prepared with acid protease. Consecutive purification methods were used for peptide isolation including ion‐exchange chromatography, size‐exclusion chromatography, and reverse‐phase high‐performance liquid chromatography. The amino acid sequence of this peptide was identified as Ile‐Ala‐Pro, and the ACE inhibitory activity (IC₅₀ value) was 2.7 μM . The hypotensive activity of Ile‐Ala‐Pro on spontaneously hypertensive rats was investigated. This peptide inhibited the hypertensive activity of angiotensin I with intravenous injection, and decreased the blood pressure significantly with intraperitoneal administration.     

Isolation,purification and characterisation of angiotensin I‐converting enzyme–inhibitory peptides derived from catfish (Clarias batrachus) muscle protein thermolysin hydrolysates

The angiotensin I‐converting enzyme (ACE)‐inhibitory activities of catfish (Clarias batrachus) muscle protein hydrolysates were investigated. Thermolytic digests of C. batrachus sarcoplasmic and myofibrillar proteins exhibited inhibitory activity towards ACE and were purified with the aim of ultrafiltration, gel filtration and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The amino acid sequences of hydrolysates with the highest ACE‐inhibitory activities were determined using electrospray quadrupole time‐of‐flight tandem mass spectrometry (ESI‐TOFQ MS/MS). The sequences of GPPP (IC₅₀ = 0.86 μm ) and IEKPP (IC₅₀ = 1.2 μm ) corresponding to the fragments 986–989 and 441–445 of myosin‐I heavy chain were identified for the sarcoplasmic and myofibrillar protein hydrolysates, respectively. Peptide GPPP exhibited a mixed‐type inhibition whereas peptide IEKPP could only bind to the active sites of ACE. The results demonstrate that hydrolysates of C. batrachus muscle proteins obtained by thermolysin may contain bioactive peptides.     

Effect of enzyme type and hydrolysis conditions on the in vitro angiotensin I‐converting enzyme inhibitory activity and ash content of hydrolysed whey protein isolate

Removal of salts from protein hydrolysate mixture on large scale is very difficult and relatively inefficient. Selecting practical proteinase system and hydrolysis conditions for the production of whey protein isolate (WPI) enzymatic hydrolysates with high angiotensin I‐converting enzyme (ACE) inhibitory activity and low ash content is very useful. The effect of alcalase, neutrase, trypsin and their combined system, i.e. alcalase‐neutrase and trypsin‐neutrase, under two different hydrolysis conditions, i.e. pH‐controlled and pH‐spontaneous drop, on the formation of ACE‐inhibitory peptides and the characteristics of WPI hydrolysate was investigated. Results showed that the ACE‐inhibitory activity of WPI hydrolysate obtained with alcalase was significantly higher than that of its trypsin or neutrase hydrolysate obtained at the same hydrolysis time by both pH‐controlled and pH‐spontaneous drop method (P < 0.05). The WPI hydrolysate obtained after 3 h incubation with alcalase plus 2 h with neutrase under pH‐spontaneous drop condition possessed the highest ACE‐inhibitory activity of 54.30% and the lowest ash content of 2.95%. This is practical as a functional ingredient in the food industry because of its high ACE‐inhibitory capability, commercial availability in large supply of alcalase and neutrase and no needing for additional desalting process.     

Preparation and purification of angiotensin‐converting enzyme inhibitory peptides from hydrolysate of shrimp (Litopenaeus vannamei) shell waste

Angiotensin I‐converting enzyme (ACE) inhibitory peptides from the shrimp shell waste (SSW) were isolated using different proteases. The orthogonal test results showed alcalase hydrolysates with ACE inhibitory activity of 67.07% under the optimal hydrolysis conditions of 60 °C hydrolysis temperature, pH = 9.5, 25 g L^?1 substrate and 4000 U g^?1 of enzyme, whereas neutral protease hydrolysates had an ACE inhibitory activity of 84.04% under the hydrolysis temperature of 50 °C at pH = 7.0 with 25 g L^?1 of substrate and in the presence of 2000 U g^?1 of enzyme. Neutral protease was more suitable for the production of ACE inhibitory peptides from SSW, where peptides with MW <5 kDa were recommended. The results of this study indicated that peptides obtained from SSW are as beneficial as antihypertension compounds in the functional food resources.     

Identification of angiotensin converting enzyme inhibitory and antioxidant peptides in a whey protein concentrate hydrolysate produced at semi‐pilot scale

Antioxidant and angiotensin converting enzyme (ACE) inhibitory peptides were identified in a 5 kDa ultrafiltration permeate of a whey protein hydrolysate generated at semi‐pilot scale. Further laboratory scale ultrafiltration of this 5 kDa permeate resulted in a 0.65 kDa permeate with antioxidant, (1.11 ± 0.074 μmol TE per mg dry weight, oxygen radical absorbance capacity, ORAC) and ACE inhibitory (ACE IC₅₀ 0.215 ± 0.043 mg mL^?1) activities. Semi‐preparative (SP) reverse phase high‐performance liquid chromatography (RP‐HPLC) of the 0.65 kDa permeate resulted in a fraction (SP_F3) with a 4.4‐fold increase in ORAC activity (4.83 ± 0.45 μmol TE mg dry weight) and a 1.3‐fold increase in ACE inhibitory activity (84.35 ± 1.36% inhibition when assayed at 0.28 mg mL^?1). Peptides within SP_F3 were identified using UPLC‐ESI‐MS/MS. Met‐Pro‐Ile had the highest ORAC activity (205.75 ± 12.08 μmol TE per mmol peptide) while Met‐Ala‐Ala and Val‐Ala‐Gly‐Thr had the highest ACE inhibitory activities (IC₅₀:515.50 ± 1.11 and 610.30 ± 2.41 μm , respectively).     

Angiotensin I‐converting enzyme inhibitory activity of protein hydrolysates prepared from three freshwater carps (Catla catla,Labeo rohita and Cirrhinus mrigala) using Flavorzyme

Fish protein hydrolysates from three freshwater carps, Catla catla, Labeo rohita and Cirrhinus mrigala with different degree of hydrolysis (DH) (5%, 10%, 15% and 20%), were prepared using Flavorzyme enzyme and designated as HCF, HRF and HMF, respectively. The angiotensin I‐converting enzyme (ACE) inhibitory activity of hydrolysates was found to vary from 43 ± 2% to 71 ± 3%. Based on ACE inhibitory activity, HRF with DH‐15% was taken up for further study. The mode of ACE activity inhibition by HRF‐DH 15% was mixed type as revealed by Lineweaver–Burk plot. Sequential digestion of HRF‐DH 15% using pepsin and pancreatin decreased the ACE inhibitory activity from 76% to 63%. Partial purification of HRF‐DH 15% by size exclusion chromatography gave three different fractions designated as F‐1, F‐2 and F‐3 with the molecular mass in the range of 6456–407 Da. Fraction 2 had significantly higher ACE inhibitory activity than the other fractions.     

Angiotensin I‐converting enzyme inhibitory peptides in skim milk fermented with Lactobacillus helveticus 130B4 from camel milk in Inner Mongolia,China

BACKGROUND: Angiotensin I‐converting enzyme (ACE) is a dipeptidyl carboxypeptidase associated with the regulation of blood pressure. ACE inhibition results in a lowering of blood pressure. Lactic acid bacteria are known to produce ACE inhibitors during fermentation. Fermented camel milk is the main traditionally fermented dairy food for desert nomads. The beneficial effects of fermented camel milk, which include the prevention of such diseases and conditions as gastroenteritis, tuberculosis and hypertension, have been demonstrated experimentally. RESULTS: ACE inhibitory activity was observed in fermented milk containing Lactobacillus helveticus 130B4, a strain isolated from traditionally fermented camel milk. The peptide that inhibited ACE was purified from the fermented milk by reverse‐phase high‐performance liquid chromatography. The amino acid sequence of the peptide was identified as Ala‐Ile‐Pro‐Pro‐Lys‐Lys‐Asn‐Gln‐Asp (IC₅₀ = 19.9 µmol L^?1). The same Ala‐Ile‐Pro‐Pro‐Lys‐Lys‐Asn‐Gln‐Asp sequence was found in κ‐casein (κ‐CN) f107–115 from milk. The inhibitory activity of this nonapeptide (κ‐CN f107–115) was almost preserved even after successive digestion with pepsin, trypsin and chymotrypsin. Furthermore, the inhibitory activity of the purified peptide was completely preserved after heat treatment at 100 °C for 20 min. CONCLUSION: The fermented milk prepared with Lactobacillus helveticus 130B4 contained an ACE inhibitory peptide, κ‐CN 107–115. This fermented milk was expected to have anti‐hypertensive effect after ingestion because the peptide was stable to digestive protease and heat treatment in vitro. Copyright © 2008 Society of Chemical Industry     

Purification and characterisation of angiotensin I converting enzyme inhibitory peptides from lysozyme hydrolysates

Hen egg white lysozyme (HEWL) was hydrolysed with trypsin, papain and a combination of the two. The prepared hydrolysates exhibited ACE inhibitory activity. The hydrolysates were fractionated using ultrafiltration and reverse phase-high performance liquid chromatography (RP-HPLC). Three fractions, which showed the highest ACE inhibitory activities, were purified by RP-HPLC. They were the F₇ (from papain-trypsin hydrolysate), F₈ (from papain hydrolysate) and F₃ (from trypsin hydrolysate) fractions. The IC₅₀ values were 0.03, 0.155 and 0.23 mg/ml for F₇, F₈ and F₃, respectively. The F₇ fraction was the most potent ACE inhibitor peptide, and was composed of 12 amino acids, Phe-Glu-Ser-Asn-Phe-Asn-Thr-Gln-Ala-Thr-Asn-Arg (MW: 1428.6 Da). Lineweaver-Burk plots suggest that the F₇ peptide acts as an uncompetitive inhibitor against ACE. The kinetic parameters (K_m, V_max, and K_i) for the F₇ peptide were measured and compared to the control.     

纳豆菌蛋白酶水解玉米醇溶蛋白制备血管紧张素转化酶抑制肽

利用纳豆菌固态发酵产酶酶解玉米醇溶蛋白,酶解物经SephadexG-25分离纯化,对产物及其组分血管紧张素转化酶(ACE)抑制活性进行检测。结果表明,酶解血管紧张素转化酶的抑制活性为77.83%,经分离后组分3的抑制效果最好,可达89.4%。     

Activities and sequences of the angiotensin I‐converting enzyme (ACE) inhibitory peptides obtained from the digested lentil (Lens culinaris) globulins

The aim of this study was the identification of potentially bioaccessible ACE‐inhibitory peptides obtained by in vitro gastrointestinal digestion of lentil globulins. ACE‐inhibitory peptides were purified by ion exchange chromatography and gel filtration. After the first step of purification, three peptide fractions with potential antihypertensive properties were obtained and the highest inhibitory activity was determined for the fraction 5 (IC₅₀ = 0.02 mg mL^?1). This fraction was separated on Sephadex G10, and six peptide fractions were obtained. The peptides of fraction (5‐F) with the highest potential antihypertensive activity (IC₅₀ = 0.13 mg mL^?1) were identified using ESI‐MS/MS. The sequences of peptides were KLRT, TLHGMV and VNRLM. Based on Lineweaver–Burk plots for the fraction 5‐F, the kinetic parameters as K_m (1.24 mm ), V_max (0.012 U min^?1), K_i (0.12 mg mL^?1) and mode of inhibition were determined.     

Grass carp peptides hydrolysed by the combination of Alcalase and Neutrase: Angiotensin‐I converting enzyme (ACE) inhibitory activity,antioxidant activities and physicochemical profiles

In this study, grass carp peptides were prepared by enzymatic hydrolysis of grass carp protein using the combination of Alcalase and Neutrase, and angiotensin‐I converting enzyme (ACE) inhibitory activity in vitro, antihypertensive activity in vivo, antioxidant activities, and physicochemical properties of peptides achieved from grass carp protein were characterised after ultrafiltration and desalted processes using mixed ion exchange resins. The purified peptides exhibited strong ACE inhibitory activity (IC₅₀ = 105 μg mL^?1), antihypertensive activity with the maximal drop for systolic blood pressure (SBP) of 43 mmHg at a dosage of 100 mg per kg body weight in spontaneously hypertensive rat (SHR), and antioxidant activities indicated by thiobarbituric acid‐reactive substance values in a liposome‐oxidising system, radical‐scavenging activity and chelation of metal ions (Fe²⁺). The molecular weight of peptides was <1000 Da. Compared to grass carp protein, the peptides separated from enzymatic hydrolysates possessed similar amino acid compositions, but contained higher concentrations of essential amino acids. Moreover, the peptides exhibited excellent solubility at a wide range of pH values from 2 to 10, and lower apparent viscosity than the protein. The peptides separated from enzymatic hydrolysates might be used as a promising ingredient in antihypertensive functional foods and nutraceuticals.     

Angiotensin‐I‐Converting Enzyme Inhibitory Activities and In Vivo Antihypertensive Effects of Sardine Protein Hydrolysate

In our previous study, an antihypertensive protein hydrolysate was prepared from sardine. This study aimed to investigate the composition of sardine protein hydrolysate (SPH) and it's in vivo antihypertensive effect. SPH was separated sequentially with ultrafiltration and size exclusion chromatography. Fractions with high angiotensin‐I‐converting enzyme (ACE) inhibitory activity were further analyzed with RP‐HPLC and amino acids analysis. Then, SPH was individually oral administrated to spontaneously hypertensive rats (SHR) and normotensive wistar kyoto rats (WKY) for 4 wk. After treatment, the systolic blood pressure (SBP), organ index, oxidative status, serum ACE activity, and serum angiotensin‐II (ANG‐II) of all the rats were determined. According to the separation and analysis results, 3 main fractions with high ACE‐inhibitory activity were obtained with different amino acids composition. The animal experiments results showed that SPH could significantly reduce SBP (P < 0.05 or P < 0.01) within 4 h after a single oral administration. After a chronic oral administration, a steady state of SBP in SHR rats was attained. The heart weight index and left ventricular weight index were significantly reduced (P < 0.05) in SPH‐treated SHR rats. The malonyldialdehyde (MDA) levels in organs and serum, serum ACE activity, and serum ANG‐II concentration in SPH‐treated SHR rats were significantly lowered (P < 0.05 or P < 0.01) as compared to their controls. Meanwhile there was no significant effect (P > 0.05) on those parameters in WKY rats. These results indicate that SPH can decrease blood pressure in SHR rats and not in WKY rats.     

Enhancement of angiotensin I converting enzyme inhibitory activity and improvement of the emulsifying and foaming properties of corn gluten hydrolysate using ultrafiltration membranes

In this study, we attempted to enhance angiotensin I converting enzyme (ACE) inhibitory activity using an ultrafiltration (UF) membrane and to improve the emulsifying and foaming properties of fractions. ACE inhibitory activities of the corn gluten hydrolysate prepared by Flavourzyme had the same trend as protein contents and the IC₅₀ value was 0.18 mg solid after 8 h hydrolysis. The hydrolysate was separated by using two kinds of UF membrane (10 and 5 kDa cut-off membranes) and three fractions, >10 kDa, 5–10 kDa, and <5 kDa, were obtained. The yields of these three fractions were 58.3%, 27.2% and 14.5% as dry matter, respectively. The IC₅₀ value (the concentration of ACE inhibitor required to inhibit 50% of the ACE activity under specific conditions) of the <5 kDa fraction was 0.05 mg solid and was approximately fourfold that of the original, 5–10, and >10 kDa fractions. The average hydrophobicity of the <5 kDa fraction was slightly lower than that of the others according to the hydrophobicity index of Nozaki and Tanford, but according to the Krigbaum and Meirovitch index, that of the <5 kDa fraction was slightly higher than those of the others, showing there might be no significant differences among the fractions. The emulsifying activity and emulsion stability in the <5 kDa fraction was higher than those in the other fractions. Two fractions, <5 kDa and >10 kDa, showed poor foaming capacity at below pH 6.0. The foam stabilities of two fractions (<5 kDa and >10 kDa) increased with increasing pH, but that of 5–10 kDa decreased with increasing pH. The UF treatment was an effective method for the enhancement of ACE inhibitory activity with improvement of functional properties.     

Proteolytic and angiotensin‐converting enzyme‐inhibitory activities of selected probiotic bacteria

This study was carried out to examine the proteolytic and angiotensin‐converting enzyme (ACE‐I) activities of probiotic lactic acid bacteria (LAB) as influenced by the type of media, fermentation time, strain type and media supplementation with a proteolytic enzyme (Flavourzyme^®). Lactobacillus casei (Lc210), Bifidobacterium animalis ssp12 (Bb12), Lactobacillus delbrueckii subsp. bulgaricus (Lb11842) and Lactobacillus acidophilus (La2410) were grown in 12% of reconstituted skim milk (RSM) or 4% of whey protein concentrates (WPC‐35) with or without combination (0.14%) of Flavourzyme^® for 12 h at 37 °C. All the strains were able to grow in both media depending on type of strains used and fermentation time. All the strains showed higher proteolytic activity and produced more antihypersensitive peptides when grown in RSM medium at 12 h, when compared to WPC. Combination with Flavourzyme^® also increased LAB growth and proteolytic and ACE‐I activities. Of the four strains used, Bb12 and La2410 outperformed Lc210 and Lb11842. The highest ACE‐I activity and proteolytic activity were found in B. animalis ssp12 combined with Flavourzyme^®. Flavourzyme^® led to an increase in the production of bioactive peptides with ACE‐I activity during 12 h at 37 °C.     

Isolation and characterisation of a novel angiotensin I‐converting enzyme‐inhibitory peptide derived from douchi,a traditional Chinese fermented soybean food

BACKGROUND: Douchi, a traditional fermented soybean food, has recently attracted a great deal of attention owing to its superior physiological activity. In the present study the angiotensin I‐converting enzyme (ACE)‐inhibitory activity of typical douchi procured from various regions of China was analysed. An ACE‐inhibitory peptide derived from the most potent douchi was also isolated and characterised. The pattern of ACE inhibition and resistance to hydrolysis by gastrointestinal proteases of this peptide are described. RESULTS: ACE‐inhibitory activities were detected in all douchi samples, with IC₅₀ values ranging from 0.204 to 2.011 mg mL^?1. Among the douchi samples, a Mucor‐type douchi exhibited the most potent ACE‐inhibitory activity (IC₅₀ = 0.204 mg mL^?1). A novel ACE‐inhibitory peptide was then isolated from this Mucor‐type douchi using ultrafiltration followed by Sephadex G‐25 column chromatography and reverse phase high‐performance liquid chromatography. The amino acid sequence of the purified peptide was identified by Edman degradation as His‐Leu‐Pro (IC₅₀ = 2.37 µmol L^?1). The peptide is a competitive inhibitor and maintained its inhibitory activity even after incubation with some gastrointestinal proteases. CONCLUSION: The present study shows that peptides derived from soybean fermentation during douchi processing could be the main contributor to the ACE‐inhibitory activity observed. Copyright © 2009 Society of Chemical Industry     

Effect of seaweed flakes addition on the development of bioactivities in functional Camembert‐type cheese

The effect of adding Palmaria palmata or Saccharina longicruris to Camembert‐type cheese on both the antioxidant capacity (ORAC) and Angiotensin I‐converting enzyme (ACE)‐inhibitory activity has been studied with the aim of developing a functional food. The nutritional composition showed that P. palmata had the highest total protein and carbohydrate contents while S. longicruris demonstrated the highest total fibre and minerals contents. The bioactivities determined in the S. longicruris soluble extract were the highest. Three cheese model curds were studied: cheese control (CC), 2% of P. palmata (C2PP) and 2% of S. longicruris (C2SL). During ripening (20 days), the curd pH of all treatments was significantly similar, as their ORAC values, starting at 0 and reaching 41.28 mmol TE g^?1. The ACE‐inhibitory activity of the three treatments was significantly similar, at day 0 (13.20%) and day 20 (58.27%). The feasibility of including these two seaweeds into Camembert‐type cheeses was validated through the adequate development of bioactivities during ripening supporting promising food applications.