Enzyme Inhibitory Peptides Research Articles

Molecular interactions, bioavailability, and cellular mechanisms of angiotensin-converting enzyme inhibitory peptides.

Food-derived angiotensin-converting enzyme inhibitory (ACEi) peptides have gained substantial interest as potential alternatives to synthetic drugs in the management of hypertension. Peptide size and sequence are two critical factors that determine their potency, bioavailability, and cellular mechanisms. Molecular interaction studies between ACE and ACEi peptides support that potent ACEi peptides are generally composed of hydrophobic, positively charged, and aromatic or cyclic amino acid residues at the third, second, and first position from the C-terminus, respectively. Small peptides containing N-terminal Tyr and/or C-terminal Pro could improve their stability against enterocyte peptidases, thus their bioavailability. Different ACEi peptides can reduce aberrant cellular proliferation, excessive inflammation, and oxidative stress but through different mechanisms. Further understanding the structure-activity-bioavailability relationships will help design novel potent ACEi peptides with improved bioavailability and in vivo efficacy. PRACTICAL APPLICATIONS: ACEi peptides have the potential for uses as functional food ingredients against hypertension.

Lactobacillus helveticus LH-B02 favours the release of bioactive peptide during Prato cheese ripening

Adjunct cultures are traditionally used in cheesemaking with the aim of improving the flavour development of ripened cheeses. Additionally, different adjunct cultures are recognised for the ability to produce enzymes with specificity to hydrolyse caseins, leading to the release of bioactive peptides. The objective of this study was to evaluate the effect of the addition of the adjunct culture Lactobacillus helveticus LH-B02 on the proteolysis and peptide profile of Prato cheese during ripening. The addition of the adjunct culture affected the Prato cheese peptide profile and favoured the release of peptides from β-casein and, in particular, the angiotensin-converting enzyme-inhibitory peptide β-CN (f194–209) (m/z 1718), which was the most important variable for samples discrimination after 60 d of ripening.

Discovery and quantification of bioactive peptides in fermented cucumber by direct analysis IR-MALDESI mass spectrometry and LC-QQQ-MS

Bioactive peptides have been identified in lactic acid bacteria fermented foods including cultured milk, sourdough, and cured meats; however, their presence has not been investigated in fermented vegetables. In this study, infrared, matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry (MS) was employed to identify bioactive peptides in fermented cucumber. Natural and starter culture fermented cucumbers were prepared in triplicate in sodium chloride brines and compared to acidified cucumbers. Putative matches of known food-derived bioactive peptides were identified by direct analysis using IR-MALDESI-MS. Peptides were confirmed by IR-MALDESI MS/MS and quantified by LC-MS/MS. Three angiotensin converting enzyme (ACE) inhibitory peptides, IPP (0.42–0.49 mg/kg), LPP (0.30–0.33 mg/kg), and VPP (0.32–0.35 mg/kg) were formed in fermented cucumbers. A fourth ACE inhibitory peptide, KP (0.93–1.5 mg/kg), was enhanced 3–5 fold in fermented cucumbers compared with acidified cucumbers. This work demonstrates that lactic acid bacteria fermentation can enhance bioactive peptide content in vegetables.

Processing Optimization and Characterization of Angiotensin-Ι-Converting Enzyme Inhibitory Peptides from Lizardfish (Synodus macrops) Scale Gelatin.

Hypertension can cause coronary heart disease. Synthetic angiotensin-converting enzyme (ACE) inhibitors are effective antihypertensive drugs but often cause side effects. The aim of this study was to prepare potential ACE inhibitors from scales. Gelatin was extracted from lizardfish scales. Then, scale gelatin was enzymolyzed to prepare ACE inhibitory peptides using response surface methodology. Proteolytic conditions after optimization were as follows: pH 7.0, enzyme substrate ratio 3.2%, temperature 47 °C, and proteolysis lasting 2 h and 50 min. The experimental ACE inhibitory activity under optimal conditions was 86.0 ± 0.4%. Among the 118 peptides identified from gelatin hydrolysates, 87.3% were hydrophilic and 93.22% had a molecular weight <2000 Da. Gelatin peptides had high stability upon exposure to high temperature and pH as well as gastrointestinal tract enzymes. Gelatin peptides showed an antihypertensive effect in spontaneously hypertensive rats at a dosage of 2 g/kg in the long-term experiments. A new ACE inhibitory peptide was isolated from gelatin hydrolysates, and was identified as AGPPGSDGQPGAK with an IC50 value of 420 ± 20 μM. In this way, ACE inhibitory peptides derived from scale gelatin have the potential to be used as healthy ACE-inhibiting drug raw materials.

The application of the Plackett-Burman design in investigating ACE inhibitory peptide-producing conditions and media for Lactobacillus bulgaricus LB6.

The intake of angiotensin converting enzyme (ACE, EC 3.4.15.1) inhibitory peptides from food has become a promising method for treating hypertension. Lactic acid producing bacteria are widely used to produce ACE inhibitors during fermentation. Several factors which promote the production of ACE inhibitory peptides in Lacto- bacillus bulgaricus LB6 (inoculum size, incubation temperature, goat&rsquo;s milk powder, sterilization time, whey powder, casein hydrolyses and calcium lactate) and media (casein peptone, soybean peptone, glucose, casein, ascorbic acid, Ca(H2PO4)2 and lactose) were investigated to optimize and increase ACE inhibitory activity during the fermentation of goat&rsquo;s milk by using a Plackett-Burman design, in accordance with previous tests. The results indicated that incubation temperature, whey powder, calcium lactate, soybean peptone,glucose and casein had significant effects on both ACE inhibition rate and viable counts. In addition, the effects of incubation temperature, whey powder, calcium lactate and glucose were found to increase ACE inhibition rate, while soybean peptone and casein caused it to decrease. This investigation of conditions which promote the production of ACE inhibitory peptides and media for LB6 was performed using a Plackett-Burman design. The results indicated that incubation tem- perature, whey powder, calcium lactate, soybean peptone, glucose and casein had a significant impact on the ACE inhibition rate and viable counts of LB6, which provide the basis and reference for further optimization.

Fermente Süt Ürünlerinin Kan Basıncını Düşürücü Etkisi

Hypertension (HTN) is a major risk factor for the development of cardiovascular&#x0D; diseases. Therefore, there is a need to lower blood pressure (BP) to reduce the&#x0D; risk of these degenerative diseases. Fermented milks contain several potential&#x0D; factors that can lower BP including calcium and microbial metabolites&#x0D; particularly the angiotension-converting enzyme (ACE) inhibitory peptides and&#x0D; γ-aminobutyric acid. &amp;nbsp;Animal studies&#x0D; clearly demonstrated the BP lowering effect of fermented milk while results&#x0D; from clinical trials were controversial due to a large number of variables that&#x0D; should be considered in clinical trials. An overview on the antihypertensive effect&#x0D; of fermented milk products is presented and discussed in this review.

Transepithelial transport and structural changes of chicken angiotensin I-converting enzyme (ACE) inhibitory peptides through Caco-2 cell monolayers

Permeability of angiotensin I-converting enzyme (ACE) inhibitory peptides (KPLLCS, ELFTT, and KPLL) identified from the in vitro gastrointestinal digest of cooked chicken breast was investigated using the Caco-2 cell model system. KPLLCS was originally the most effective ACE inhibitor (IC50 0.37 μM), but it was degraded during permeation through Caco-2 cells. Among these peptides, KPLL showed the highest permeability in intact form, and partially degraded to KP and LL, which still showed ACE inhibitory activity after permeation. ACE inhibitory activity of permeated KPLL was highest of 56%. KPLL and KP possessed a mixed inhibitor characteristic, while LL was a non-competitive inhibitor. Based on molecular docking, K at N-terminus of KPLL is a key structure contributing to ACE inhibition as it can occupy the active site of ACE. Determining the structural stability and degree of peptide transport across epithelial cell is required to confirm their potential as ACE inhibitors.

Analysis and Evaluation of the Inhibitory Mechanism of a Novel Angiotensin-I-Converting Enzyme Inhibitory Peptide Derived from Casein Hydrolysate.

Casein hydrolysates exert various biological activities, and the responsible functional peptides are being identified from them continuously. In this study, the tryptic casein hydrolysate was fractionated by an ultrafiltration membrane (3 kDa), and the peptides were identified by capillary electrophoresis-quadrupole-time-of-flight-tandem mass spectrometry. Meanwhile, in silico methods were used to analyze the toxicity, solubility, stability, and affinity between the peptides and angiotensin-I-converting enzyme (ACE). Finally, a new angiotensin-I-converting enzyme inhibitory (ACEI) peptide, EKVNELSK, derived from αs1-casein (fragment 35-42) was screened. The half maximal inhibitory concentration value of the peptide is 5.998 mM, which was determined by a high-performance liquid chromatography method. The Lineweaver-Burk plot indicated that this peptide is a mixed-type inhibitor against ACE. Moreover, Discovery Studio 2017 R2 software was adopted to perform molecular docking to propose the potential mechanisms underlying the ACEI activity of the peptide. These results indicated that EKVNELSK is a new ACEI peptide identified from casein hydrolysate.

Identification of an ACE-Inhibitory Peptide from Walnut Protein and Its Evaluation of the Inhibitory Mechanism.

In the present study, a novel angiotensin I-converting enzyme inhibitory (ACE inhibitory) peptide, EPNGLLLPQY, derived from walnut seed storage protein, fragment residues 80–89, was identified by ultra-high performance liquid chromatography electrospray ionization quadrupole time of flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) from walnut protein hydrolysate. The IC50 value of the peptide was 233.178 μM, which was determined by the high performance liquid chromatography method by measuring the amount of hippuric acid (HA) generated from the ACE decomposition substrate (hippuryl-l-histidyl-l-leucine (HHL) to assess the ACE activity. Enzyme inhibitory kinetics of the peptide against ACE were also conducted, by which the inhibitory mechanism of ACE-inhibitory peptide was confirmed. Moreover, molecular docking was simulated by Discovery Studio 2017 R2 software to provide the potential mechanisms underlying the ACE-inhibitory activity of EPNGLLLPQY.

Optimization of Nutrient Composition for Producing ACE Inhibitory Peptides from Goat Milk Fermented by Lactobacillus bulgaricus LB6.

Hypertension is a serious threat to human health and food-derived angiotensin converting enzyme (ACE; EC 3.4.15.1) inhibitory peptides can be used to regulate high blood pressure without side effects. The composition of the nutrient medium for the production of these peptides by fermenting goat milk with Lactobacillus bulgaricus LB6 was optimized to increase the ACE inhibitory activity by Box-Behnken design (BBD) of response surface methodology (RSM) in the present study. Soybean peptone, glucose, and casein had significant effects on both ACE inhibition rate and viable counts of L. bulgaricus LB6 during incubation. The results showed that the maximum values of ACE inhibition rate and viable counts for L. bulgaricus LB6 were reaching to 86.37 ± 0.53% and 8.06 × 107 under the optimal conditions, which were 0.35% (w/w) soybean peptone, 1.2% (w/w) glucose, and 0.15% (w/w) casein. The results were in close agreement with the model prediction. The optimal values of the medium component concentrations can be a good reference for obtaining ACE inhibitory peptides from goat milk.

Characterization of antioxidant and angiotensin-converting enzyme inhibitory peptides derived from cauliflower by-products by multidimensional liquid chromatography and bioinformatics

In the present paper, bioactive peptides from cauliflower by-products (leaves and stems) were investigated. Alcalase® protein hydrolysis time and pH conditions were optimized, then the hydrolysates were fractionated by preparative RP-HPLC into 12 fractions. Each fraction was tested for the ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity and for ACE (angiotensin-converting enzyme) inhibitor activity. The peptides in the most active fractions were identified by peptidomics technologies and screened for bioactivity by the use of bioinformatics. For ACE-inhibitor activity, two peptides were synthetized, APYDPDWYYIR and SKGFTSPLF, which provided an EC50 value of 2.59 μmol L−1 and 15.26 μmol L−1, respectively. For the ABTS radical scavenging activity, SKGFTSPLF and LDDPVFRPL were tested, and provided an EC50 of 10.35 μmol L−1 and 8.29 μmol L−1, respectively. For the DPPH radical scavenging activity, SKGFTSPLF and LRAPPGWTGR were tested and provided an EC50 of 8.2 μmol L−1 and 5.26 μmol L−1, respectively.

Novel angiotensin I-converting enzyme inhibitory peptides from soybean protein isolates fermented by Pediococcus pentosaceus SDL1409

In this study, Pediococcus pentosaceus SDL1409 was used to ferment soybean protein isolates for 48 h at 37 °C. Low molecular weight peptides (≤7 KDa) from the fermented sample showed strong angiotensin I-converting enzyme inhibitory ability of 65.1 ± 0.78% (IC50 = 0.123 ± 0.02 mg protein/ml). The low molecular weight peptides were identified using liquid chromatography-electrospray ionization quantitative time-of-flight mass spectrometry analysis. On the basis of peptide abundance and common structural features, four peptides were selected and chemically synthesized. The peptides namely EDEVSFSP, SRPFNL, RSPFNL and ENPFNL showed strong IC50 values of 0.571 ± 0.12, 0.131 ± 0.02, 0.811 ± 0.05 and 0.287 ± 0.07 mg/mL respectively. EDEVSFSP was fairly resistant to gastrointestinal enzyme digestion. The presence of P/FNL, the N-terminal sequence as well as the position of amino acids in the peptides were essential for ACE inhibition. Our data demonstrate that Pediococcus pentosaceus SDL1409 could be useful for developing antihypertensive functional foods from soybean proteins.

Identification and molecular docking study of novel angiotensin-converting enzyme inhibitory peptides from Salmo salar using in silico methods.

In order to circumvent some challenges of the classical approach, the in silico method has been applied to the discovery of angiotensin-converting enzyme (ACE) inhibitory peptides from food proteins. In this study, some convenient and efficient in silico tools were utilized to identify novel ACE inhibitory peptides from Salmo salar. Collagen from Salmo salar was digested in silico into hundreds of peptides. Results revealed that tetrapeptides PGAR and IGPR showed potent ACE inhibitory activity, with IC50 values of 0.598 ± 0.12 and 0.43 ± 0.09 mmol L-1 , respectively. The molecular docking result showed that PGAR and IGPR interact with ACE mostly via hydrogen bonds and attractive charge. Peptide IGPR interacts with Zn+ at the ACE active site, showing high inhibitory activity. Interaction with Zn+ in ACE may lead to higher inhibitory activity of peptides, and Pi interactions may promote the effect of peptides on ACE. The in silico method can be an effective method to predict potent ACE inhibitory peptides from food proteins. © 2018 Society of Chemical Industry.

Quantitative Structure-Activity Relationship Modeling Coupled with Molecular Docking Analysis in Screening of Angiotensin I-Converting Enzyme Inhibitory Peptides from Qula Casein Hydrolysates Obtained by Two-Enzyme Combination Hydrolysis.

In this study, Qula casein derived from yak milk casein was hydrolyzed using a two-enzyme combination approach, and high angiotensin I-converting enzyme (ACE) inhibitory activity peptides were screened by quantitative structure-activity relationship (QSAR) modeling integrated with molecular docking analysis. Hydrolysates (<3 kDa) derived from combinations of thermolysin + alcalase and thermolysin + proteinase K demonstrated high ACE inhibitory activities. Peptide sequences in hydrolysates derived from these two combinations were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). On the basis of the QSAR modeling prediction, a total of 16 peptides were selected for molecular docking analysis. The docking study revealed that four of the peptides (KFPQY, MPFPKYP, MFPPQ, and QWQVL) bound the active site of ACE. These four novel peptides were chemically synthesized, and their IC50 was determined. Among these peptides, KFPQY showed the highest ACE inhibitory activity (IC50 = 12.37 ± 0.43 μM). Our study indicated that Qula casein presents an excellent source to produce ACE inhibitory peptides.

Considering the potential of Lactobacillus rhamnosus for producing Angiotensin I-Converting Enzyme (ACE) inhibitory peptides in fermented camel milk (Indian breed)

Abstract In this study, fermented camel milk was prepared using proteolytic Lactobacillus rhamnosus MTCC 5945 (NS4), which was analyzed for X-prolyl-dipeptidyl aminopeptidase activity and Angiotensin I-Converting Enzyme (ACE) inhibitory activity. The growth conditions (inoculation rate and incubation time) for the production of peptides were optimized using OPA method. Fractionated 3 kDa, 5 kDa and 10 kDa permeate and retentate samples were further analyzed for ACE-inhibitory activity under the optimized growth conditions. Furthermore, 3 kDa and 10 kDa permeate with highest ACE-inhibitory activities and highest percentage of peptides production were subjected to liquid chromatography mass spectrometry and peptide identification using MASCOT software. Novel peptides were identified from fermented camel milk using homology sequence searching in BlastP (NCBI), and Protein information resource database (PIR). The novelty (ACE-inhibitory activity or Antihypertensive activity) of peptides was also confirmed using novelty search in the database of antihypertensive peptides (AHTPDB).

Yak milk casein as potential precursor of angiotensin I-converting enzyme inhibitory peptides based on in silico proteolysis

Yak milk casein was selected as a potential precursor of bioactive peptides based on in silico analysis. Most notable among these are the angiotensin I-converting enzyme (ACE) inhibitory peptides. First, yak milk casein has high homology with cow milk casein by homologous analysis. The potential of yak milk casein for the releasing bioactive peptides was evaluated by determining the frequency of occurrence of fragments with a given activity. Through the BIOPEP database analysis, there are many bioactive peptides in yak milk casein sequences. Then, an in silico proteolysis using single or combined enzymes to obtained ACE inhibitory peptides was investigated. Cytotoxicity analysis using the online toxic prediction tool ToxinPred revealed that all in silico proteolysis derived ACE inhibitory peptides are non-cytotoxic. Overall, the present study highlights a in silico proteolysis approach to assist the yak milk casein releasing ACE inhibitory peptides and provides a guidance for the actual hydrolysis of proteins for the production of bioactive peptides.

High angiotensin-I converting enzyme (ACE) inhibitory activity of Alcalase-digested green soybean (Glycine max) hydrolysates

As a protein-rich, underutilized crop, green soybean could be exploited to produce hydrolysates containing angiotensin-I converting enzyme (ACE) inhibitory peptides. Defatted green soybean was hydrolyzed using four different food-grade proteases (Alcalase, Papain, Flavourzyme and Bromelain) and their ACE inhibitory activities were evaluated. The Alcalase-generated green soybean hydrolysate showed the highest ACE inhibitory activity (IC50: 0.14 mg/mL at 6 h hydrolysis time) followed by Papain (IC50: 0.20 mg/mL at 5 h hydrolysis time), Bromelain (IC50: 0.36 mg/mL at 6 h hydrolysis time) and Flavourzyme (IC50: 1.14 mg/mL at 6 h hydrolysis time) hydrolysates. The Alcalase-generated hydrolysate was profiled based on its hydrophobicity and isoelectric point using reversed phase high performance liquid chromatography (RP-HPLC) and isoelectric point focusing (IEF) fractionators. The Alcalase-generated green soybean hydrolysate comprising of peptides EAQRLLF, PSLRSYLAE, PDRSIHGRQLAE, FITAFR and RGQVLS, revealed the highest ACE inhibitory activity of 94.19%, 99.31%, 92.92%, 101.51% and 90.40%, respectively, while their IC50 values were 878 μM, 532 μM, 1552 μM, 1342 μM and 993 μM, respectively. It can be concluded that Alcalase-digested green soybean hydrolysates could be exploited as a source of peptides to be incorporated into functional foods with antihypertensive activity.

Production and fermentation characteristics of angiotensin-I-converting enzyme inhibitory peptides of goat milk fermented by a novel wild Lactobacillus plantarum 69

Food derived angiotensin-I-converting enzyme (ACE)-inhibitory peptides are normally from products fermented by Lactobacillus helveticus but seldom by other lactic acid bacteria. In this study, a novel wild strain L. plantarum 69 was identified genetically and applied to ferment goat milk with high ACE-inhibitory activity, flourished bacterial population, and ideal flavor. The fermentation condition was optimized with the aid of response surface methodology (RSM). The optimal fermentation condition were temperature of 35 °C, CaCl2 concentration of 0.07%, and Tween-80 concentration of 0.04%. The ACE-inhibitory activity of the fermented product reach 88.91% which was approximately close to the predicted 91.68%. Purification was operated by ultrafiltration, macroporous resin DA201-C, gel chromatography, and reverse-phase high performance liquid chromatography (RP-HPLC), and finally exhibited the ACE-inhibitory activity of 91.62%. Goat milk fermented by L69 successfully survived in gastrointestinal tract and maintained high ACE-inhibitory activity.

Capillary liquid chromatography-ion trap-mass spectrometry methodology for the simultaneous quantification of four angiotensin-converting enzyme-inhibitory peptides in Prunus seed hydrolysates

Prunus genus fruit seeds are sources of highly angiotensin-I-converting enzyme (ACE)‐inhibitory peptides. The presence of peptides IYSPH, IYTPH, IFSPR, and VAIP seems to be related to this activity but no previous work has demonstrated the direct relationship between the concentration of these peptides and the antihypertensive activity of hydrolysates. This work describes the development of a method for the quantification of these peptides in Prunus seeds hydrolysates based on capillary liquid chromatography‐IT-MS/MS. The analytical characteristics of the method were evaluated through the study of the linearity, LOD, LOQ, presence of matrix interferences, precision, and recovery. The developed methodology was applied to the determination of the four peptides in seed hydrolysates from different Prunus genus fruits: peaches (7 varieties), plums (2 varieties), nectarines (3 varieties), apricots (2 varieties), cherry, and paraguayo. Peaches and plums seed hydrolysates yielded the highest concentrations of these peptides while paraguayo one showed the lowest concentrations. A high correlation between peptides concentrations was demonstrated suggesting that the four peptides could be released from the same seed proteins.

Antihypertensive Effects, Molecular Docking Study, and Isothermal Titration Calorimetry Assay of Angiotensin I-Converting Enzyme Inhibitory Peptides from Chlorella vulgaris.

The aim of this work is to explore angiotensin I-converting enzyme (ACE) inhibitory peptides from Chlorella vulgaris (C. vulgaris) and discover the inhibitory mechanism of the peptides. After C. vulgaris proteins were gastrointestinal digested in silico, several ACE inhibitory peptides with C-terminal tryptophan were screened. Among them, two novel noncompetitive ACE inhibitors, Thr-Thr-Trp (TTW) and Val-His-Trp (VHW), exhibited the highest inhibitory activity indicated by IC50 values 0.61 ± 0.12 and 0.91 ± 0.31 μM, respectively. Both the peptides were demonstrated stable against gastrointestinal digestion and ACE hydrolysis. The peptides were administrated to spontaneously hypertensive rats (SHRs) in the dose 5 mg/kg body weight, and VHW could decrease 50 mmHg systolic blood pressure of SHRs (p < 0.05). Molecular docking displayed that both TTW and VHW formed six hydrogen bonds with active site pockets of ACE. Besides, isothermal titration calorimetry assay discovered that VHW could form more stable complex with ACE than TTW. Therefore, VHW was an excellent ACE inhibitor.