Potent Angiotensin I-converting Enzyme Inhibitory Activity Research Articles

Screening potential hypertensive peptides using two consecutive bioassay-guided SPE fractionations and identification of an ACE inhibitory peptide, DHSTAVW (DW7), derived from pearl garlic protein hydrolysate

The pearl garlic (Allium sativum L.) protein (PGP) was digested using pepsin, trypsin, α-chymotrypsin, thermolysin, and simulated gastrointestinal digestion. The α-chymotrypsin hydrolysate showed the highest angiotensin-I-converting enzyme inhibitory (ACEI) activity, with an IC50 value of 190.9 ± 11 µg/mL. A reversed-phase C18 solid-phase extraction (RP-SPE) cartridge was used for the first fractionation, and the S4 fraction from RP-SPE showed the most potent ACEI activity (IC50 =124.1 ± 11 3 µg/mL). The S4 fraction was further fractionated using a hydrophilic interaction liquid chromatography SPE (HILIC-SPE). The H4 fraction from HILIC-SPE showed the highest ACEI activity (IC50 =57.7 ± 3 µg/mL). Four ACEI peptides (DHSTAVW, KLAKVF, KLSTAASF, and KETPEAHVF) were identified from the H4 fraction using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and their biological activities were appraised in silico. Among the identified α-chymotryptic peptides, DHSTAVW (DW7), derived from I lectin partial protein, exhibited the most potent ACEI activity (IC50 value of 2.8 ± 0.1 µM). DW7 was resistant to simulated gastrointestinal digestion, and it was classified as a prodrug-type inhibitor according to the preincubation experiment. The inhibition kinetics indicated that DW7 was a competitive inhibitor, which was rationalized by the molecular docking simulation. The quantities of DW7 in 1 mg of hydrolysate, S4 fraction, and H4 fraction were quantified using LC-MS/MS to give 3.1 ± 0.1, 4.2 ± 0.1, and 13.2 ± 0.1 µg, respectively. The amount of DW7 was significantly increased by 4.2-fold compared with the hydrolysate, which suggested that this method is efficient for active peptide screening.

Preparation, Purification and Characterization of Antibacterial and ACE Inhibitory Peptides from Head Protein Hydrolysate of Kuruma Shrimp, Marsupenaeus japonicus.

Kuruma shrimp (Marsupenaeus japonicus) heads, as the main by-product of the seafood processing industry, are rich in underutilized high-quality protein. After papain hydrolysis at 50 °C for 4 h, the protein hydrolysate of shrimp heads was found to show notable antibacterial and angiotensin I-converting enzyme (ACE) inhibitory activities. After purification using two stages of revered-phase high-performance liquid chromatography (RP-HPLC), the antibacterial peptide VTVP and the ACE inhibitory peptide ARL/I were successfully identified from most active fractions by LC-MS/MS. Peptide VTVP was a desirable hydrophobic peptide, with a MIC value in the range from 1.62 to 8.03 mM against all tested pathogens. Peptide ARL/I exhibited potent ACE inhibitory activity, with an IC50 value of 125.58 µM, and was found to be a competitive inhibitor based on the Lineweaver-Burk plot. Moreover, the result of the molecular docking simulation indicated that the interaction binding between ARL/I and ACE was mainly stabilized by hydrogen bonds, as well as forming a coordinate bond with the Zn2+ site. The purified peptides did not show hemolytic activity toward rabbit erythrocytes. To sum up, the bioactive peptides isolated from shrimp heads could be applicable for food or pharmaceutical areas as promising ingredients.

Synthesis and evaluation of new phosphonated δ‐lactam and glutarimide derivatives as angiotensin converting enzyme inhibitors

AbstractAn efficient synthesis of new γ,δ‐insaturated δ‐lactam and glutarimide derivatives bearing a phosphonomethyl group from a common allylphosphonate precursor is described. Our approach is based on a two‐step procedure involving the preparation of phosphonated‐1,5‐ketoester and −1,5‐diester followed by an amidation–heterocyclization sequence. The first step proceeds via Michael's addition of ethyl acetoacetate and diethyl malonate on an allylphosphonate starting material. The second step consists of a base‐promoted intramolecular amidation‐cyclization sequence with primary amines, which accounts for the construction of δ‐lactams and glutarimide skeletons. We performed the evaluation of angiotensin I‐converting enzyme (ACE) inhibition using an in vitro enzyme assay on six new compounds. Five compounds showed potent ACE inhibitory activity, with IC50 values ranging from 0.02 to 0.27 mg/ml. Compared with Captopril, used as a reference drug, two new glutarimide derivatives exhibited higher efficiency ACE inhibition activity.

Antioxidant properties, phenolic and mineral profiling, assessment of angiotensin I converting enzyme (ACE) inhibitory potential of Elsholtzia communis (Collett & Hemsl.) Diels from North East India

IntroductionElsholtzia communis (Collett & Hemsl.) Diels, (family: Lamiaceae) is a wild edible plant consumed as a vegetable by many tribes of the North-Eastern states of India. The objective of our study was to explore the antioxidant capacity, phenolic and mineral composition and angiotensin-I converting enzyme (ACE) inhibitory properties of E. communis. MethodsThe leaf, stem, and root extract of E. communis were screened for their antioxidant and ACE inhibitory properties. Quantification of ten phenolics was conducted using reverse phase HPLC-DAD analysis and the five phenolics were isolated using conventional column chromatography and correlated with antioxidant activities using Pearson's coefficient. An assessment of dietary minerals present in the leaf, stem, and root of this herb was carried out using ion chromatography. ResultsThe leaf exhibited the most potent DPPH scavenging and ACE inhibitory activity with IC50 value of 20.38± 0.061 µg/mL and 20.34± 0.025 µg/mL respectively. Leaf was recognized as the most prominent source of TPC, TFC, K+, Na+, Ca2+ and Mg2+. The HPLC analysis had established the stem as a rich source of gallic acid (1.08±0.002 mg/g DW), quercetin (0.26±0.001 mg/g DW) and p-coumaric acid (0.17± 0.001 mg/g DW), while catechol (0.19±0.005 mg/g DW) and vanillic acid (0.07±0.005 mg/g DW) contents were found to be highest in the leaf. NMR and HRMS data confirmed the structures of the isolated phenolics. ConclusionThus, diverse pharmacological behavior and distinctive presence of nutraceuticals and important dietary minerals suggest that this herb may be useful in the future development of herbal formulations.

ACE Inhibitory Activity and Molecular Docking of Gac Seed Protein Hydrolysate Purified by HILIC and RP-HPLC.

Gac (Momordica cochinchinensis Spreng.) seed proteins (GSPs) hydrolysate was investigated for angiotensin I-converting enzyme (ACE) inhibitory activities. GSPs were hydrolyzed under simulated gastrointestinal digestion using a combination of enzymes, including pepsin, trypsin, and chymotrypsin. The screening of ACE inhibitory peptides from GSPs hydrolysate was performed using two sequential bioassay-guided fractionations, namely hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC). Then, the peptides in the fraction with the highest ACE inhibitory activity were identified by LC-MS/MS. The flow-through (FT) fraction showed the most potent ACE inhibitory activity when HILIC fractionation was performed. This fraction was further separated using RP-HPLC, and the result indicated that fraction 8 (RP-F8) showed the highest ACE inhibitory activity. In the HILIC-FT/RP-F8 fraction, 14 peptides were identified using LC-MS/MS analysis coupled with de novo sequencing. These amino acid chains had not been recorded previously and their ACE inhibitory activities were analyzed in silico using the BIOPEP database. One fragment with the amino acid sequence of ALVY showed a significant ACE inhibitory activity (7.03 ± 0.09 µM). The Lineweaver-Burk plot indicated that ALVY is a competitive inhibitor. The inhibition mechanism of ALVY against ACE was further rationalized through the molecular docking simulation, which revealed that the ACE inhibitory activities of ALVY is due to interaction with the S1 (Ala354, Tyr523) and the S2 (His353, His513) pockets of ACE. Bibliographic survey allowed the identification of similarities between peptides reported as in gac fruit and other proteins. These results suggest that gac seed proteins hydrolysate can be used as a potential nutraceutical with inhibitory activity against ACE.

Purification and identification of peptides with high angiotensin-I converting enzyme (ACE) inhibitory activity from honeybee pupae (Apis mellifera) hydrolysates with in silico gastrointestinal digestion

The objective was to identify peptides with ACE inhibitory activity generated from honeybee pupae hydrolysates. In addition, we simulated gastrointestinal digestion and predicted the potential cleavage sites of the identified peptides. Peptides derived from honeybee pupae were cleaved in silico by pepsin, trypsin, and chymotrypsin using the PeptideCutter tool of ExPASy. Honeybee pupae were hydrolyzed using four different proteases (Alcalase, neutrase, trypsin, and papain), and their ACE inhibitory activity and distribution of molecular weights were determined. The neutrase hydrolysates showed the highest ACE inhibitory activity. Then, the hydrolysates were isolated and purified using ultrafiltration, gel filtration chromatography, and reversed-phased high performance liquid chromatography. Three novel ACE inhibitory peptides were identified by LC–MS/MS, AVFPSIVGR, PPVLVFV, and PGKVHIT, which exhibited the most potent ACE inhibitory activity, with IC50 values of 6.64, 47.7859, and 223.869 µM, respectively. In simulated in silico gastrointestinal digestion, peptides AVPFSIVGR and PGKVHIT had the position of cleavage sites by pepsin, chymotrypsin and trypsin, and PPVLVFV was cleaved by pepsin and chymotrypsin. We hope these results support the application of honeybee pupa hydrolysates as functional ingredients in foods and pharmaceuticals.

Identification and Molecular Docking Study of a Novel Angiotensin-I Converting Enzyme Inhibitory Peptide Derived from Enzymatic Hydrolysates of Cyclina sinensis.

Marine-derived angiotensin-I converting enzyme (ACE) inhibitory peptides have shown potent ACE inhibitory activity with no side effects. In this study, we reported the discovery of a novel ACE-inhibitory peptide derived from trypsin hydrolysates of Cyclina sinensis (CSH). CSH was separated into four different molecular weight (MW) fractions by ultrafiltration. Fraction CSH-I showed the strongest ACE inhibitory activity. A peptide was purified by fast protein liquid chromatography (FPLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) and its sequence was determined to be Trp-Pro-Met-Gly-Phe (WPMGF, 636.75 Da). The Lineweaver-Burk plot showed that WPMGF was a competitive inhibitor of ACE. WPMGF showed a significant degree of stability at varying temperatures, pH, and simulated gastrointestinal environment conditions. We investigated the interaction between this pentapeptide and ACE by means of a flexible molecular docking tool. The results revealed that effective interaction between WPMGF and ACE occurred mainly through hydrogen bonding, hydrophobic interactions, and coordination bonds between WPMGF and Zn(II). In conclusion, our study indicates that a purified extract derived from Cyclina sinensis or the WPMGF peptide could potentially be incorporated in antihypertensive functional foods or dietary supplements.

Optimization and Scale-Up Preparation of Egg White Hydrolysate with Angiotensin I Converting Enzyme Inhibitory Activity.

Angiotensin I converting enzyme (ACE) plays an important role in regulation of blood pressure as it converts angiotensin I into angiotensin II (a potent vasoconstrictor). Food protein-derived ACE inhibitory peptides have been considered as a safer alternative to antihypertensive drugs. In our previous study, three ACE inhibitory peptides were characterized from egg white ovotransferrin and their antihypertensive activity has been validated in spontaneously hypertensive rats. However, it is too costly to prepare these peptides from purified egg white ovotransferrin. The aims of the study were to determine the feasibility of preparing these peptides using egg white and then to optimize the conditions of preparing egg white hydrolysate. Taguchi's method was used to design experiments for optimization, which was established as follows: substrate %, pH of thermoase, time of thermoase digestion, ratio of pepsin to substrate, pH of pepsin, temperature of pepsin, and time of pepsin digestion were 7.5%, pH 8, 90 min, 1%, pH 2.5, 55 °C, and 180 min, respectively. The ACE inhibitory activity (IC50 value) and peptide yield obtained under optimal condition were 30 ± 2 μg/mL and 77.5% ± 0.3%, respectively, which were comparable to the predicted values. Hydrolysate prepared at 150 L reactor showed comparable activity but low peptide yield. Results of this study demonstrated the feasibility of using egg white protein as the starting material to prepare a functional ingredient with potent ACE inhibitory activity.

Antioxidant and angiotensin-I converting enzyme inhibitory activities of phenolic extracts and fractions derived from three phenolic-rich legume varieties

Lentil, black soybean and black turtle bean are commonly consumed legumes of different genera, containing high phenolic contents, which are effective antioxidants and angiotensin-I converting enzyme (ACE) inhibitors. However, these legumes’ phenolic compositions and ACE inhibition ability have not been compared. Crude water extract (CE) was semi-purified (SPE) and fractionated using column chromatography. Results showed that purification and fractionation could substantially increase phenolic contents and antioxidant capacities. Heating and variety had great effect on phenolic substances, antioxidant potential and mass yield of extracts and fractions. Only crude extracts showed potent ACE inhibitory activity. Black turtle bean’s ACE inhibition potential was largely reduced by cooking. The order from low to high in terms of ACE inhibitory activity was black turtle bean < lentil < black soybean. Identification and quantification of individual phenolic compounds by UV spectroscopy and LC-MSn analysis confirmed 18, 22, and 14 compounds, respectively, for the three legumes.

Antioxidant and angiotensin I-converting enzyme inhibitory activities of northern shrimp (Pandalus borealis) by-products hydrolysate by enzymatic hydrolysis

In the present study, we investigated to the antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of the northern shrimp (Pandalus borealis) by-products (PBB) hydrolysates prepared by enzymatic hydrolysis. The antioxidant and ACE inhibitory activities of five enzymatic hydrolysates (alcalase, protamex, flavourzyme, papain, and trypsin) of PBB were evaluated by the 2, 2′-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS+) radical scavenging and superoxide dismutase (SOD)-like activities, reducing power and Li’s method for ACE inhibitory activity. Of these PBB hydrolysates, the protamex hydrolysate exhibited the most potent ACE inhibitory activity with IC50 value of 0.08 ± 0.00 mg/mL. The PBB protamex hydrolysate was fractionated by two ultrafiltration membranes with 3 and 10 kDa (below 3 kDa, between 3 and 10 kDa, and above 10 kDa). These three fractions were evaluated for the total amino acids composition, antioxidant, and ACE inhibitory activities. Among these fractions, the 10 kDa fraction, while the > 10 kDa fraction exhibited the significant reducing power than others. In addition, 3–10 kDa and > 10 kDa fractions showed the significant ACE inhibitory activity. These results suggested that the high molecular weight enzymatic hydrolysate derived from PBB could be used for control oxidative stress and prevent hypertension.

Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from cauliflower by-products protein hydrolysate

Cauliflower by-products (stems and leaves) are rich in leaf protein. Making full use of cauliflower by-products can not only alleviate the shortage of protein resources but also be beneficial to the environmental protection. This study sought to prepare and purify an angiotensin I-converting enzyme (ACE) inhibitory peptide from cauliflower by-products protein hydrolysate. Our result showed that the highest ACE inhibitory activity was observed in the Alcalase 2.4L hydrolysate among tested hydrolysates (pepsin, pancreatin, and Alcalase 2.4L). After purification, a dipeptide (Val-Trp) was identified, with a molecular weight of 304.1665Da. This peptide had a potent ACE inhibitory activity with IC50 of 31.30μM. Based on this purification process, approximately 42mg of this dipeptide was obtained from 1kg of fresh cauliflower by-products. Overall, the results suggest that cauliflower by-products protein can be used as an economical source of functional food material against hypertension.

Hydrolysis and purification of ACE inhibitory peptides from the marine microalga Isochrysis galbana

Response surface methodology (RSM) was employed to optimize enzymatic hydrolysis conditions for the production of angiotensin I-converting enzyme (ACE) inhibitory peptides from the marine microalga Isochrysis galbana. A hydrolysis temperature of 55.64 °C, a substrate concentration of 5.46 g (100 mL)−1, and a trypsin enzyme/substrate ratio (E/S) of 6.27 % were found to be optimal for obtaining the highest ACE inhibitory activity of 47.62 %. The protein hydrolysates were then separated using Sephadex G-25 column chromatography and an AKTA purifier (Inertsil ODS-3 C18, 10 × 250 mm). Using tandem mass spectrometry, the active form of the purified peptide with the most potent ACE inhibitory activity was identified as Tyr-Met-Gly-Leu-Asp-Leu-Lys, with an IC50 of 36.1 μM. This study improves our understanding of the ACE inhibitory properties of I. galbana protein hydrolysate and may be useful in further identification of ACE inhibitory peptides in other marine algae.

Evaluation and Identification of Potent Angiotensin-I Converting Enzyme Inhibitory Peptide Derived from Dwarf Gulper Shark (C entrophorus atromarginatus )

Dwarf gulper shark (Centrophorus atromarginatus) lives in tropical to temperate waters and has a plenty of squalene in the liver. On the other hand, its muscle is an unused resource in Japan because of high urea content. Here, we tried to find the new procedure for the preparation of angiotensin-I converting enzyme (ACE) inhibitory hydrolysate from this shark. Nine hydrolysates were prepared using food-processing proteases. Among them, three hydrolysates produced by Protease P, Papain and Thermoase showed the potent ACE inhibitory activity (The IC50 values: 172.2, 144.2 and 84.2 μg/mL, respectively). From Thermoase hydrolysate, Val-Trp was found as the most potent inhibitory peptide (IC50 value: 3.3 μg/mL). The mechanism of inhibition of Val-Trp for ACE was competitive. Val-Trp also exhibited antioxidant activities, suggesting the efficacy as bifunctional peptide against hypertension. This study showed the new possibility of the usage of dwarf gulper shark as material for healthy food. Practical Applications This study introduces processing techniques for potent ACE inhibitory hydrolysate from dwarf gulper shark muscle by using food-processing proteases. This hydrolysate shows low bitterness, which is suitable for the usage as functional food. The strongest ACE inhibitory peptide in this hydrolysate also possessed antioxidant activity, suggesting that the hydrolysate would be useful for hypertension acting as bifunctional (ACE inhibitory and antioxidative). Results of this study will contribute to utilize dwarf gulper shark muscle, a nonutilized resource in Japan.

Novel angiotensin I-converting enzyme inhibitory peptides derived from edible mushroom Agaricus bisporus (J.E. Lange) Imbach identified by LC–MS/MS

Angiotensin I-converting enzyme (ACE) inhibitors derived from foods are valuable auxiliaries to agents such as captopril. Eight highly functional ACE inhibitory peptides from the mushroom, Agaricus bisporus, were identified by LC–MS/MS. Among these peptides, the most potent ACE inhibitory activity was exhibited by AHEPVK, RIGLF and PSSNK with IC50 values of 63, 116 and 129μM, respectively. These peptides exhibited high ACE inhibitory activity after gastrointestinal digestion. Lineweaver–Burk plots suggested that AHEPVK and RIGLF act as competitive inhibitors against ACE, whereas PSSNK acts as a non-competitive inhibitor. Mushrooms can be a good component of dietary supplement due to their readily available source and, in addition, they rarely cause food allergy. Compared to ACE inhibitory peptides isolated from other edible mushrooms, AHEPVK, RIGLF and PSSNK have lower IC50 values. Therefore, these peptides may serve as an ideal ingredient in the production of antihypertensive supplements.

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

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

Angiotensin I converting enzyme inhibitory activity and antihypertensive effect in spontaneously hypertensive rats of cobia (Rachycentron canadum) head papain hydrolysate

Cobia head protein hydrolysate (CHPH) with angiotensin I converting enzyme (ACE) inhibitory activity was prepared with papain. The 3 kDa ultrafiltration filtrate CHPH-IV of the hydrolysate exerted a potent ACE inhibitory activity with IC50 being 0.24 mg/mL. The fractions with molecular weight located between 1749 Da and 173 Da represented up 66.96% of CHPH-IV, and those between 494 Da and 173 Da represented up 31.37% of CHPH-IV. It was found that the ACE inhibitory activity of CHPH-IV was intensified from IC50 0.24 mg/mL to 0.17 mg/mL after incubation with gastrointestinal proteases. The CHPH-IV significantly decreased the systolic blood pressure in a dose-dependent manner after oral administration to spontaneously hypertensive rats (SHR) at dose of 150 mg/kg, 600 mg/kg and 1200 mg/kg body weight. These results suggested that CHPH-IV from cobia head protein hydrolysate by papain could serve as a source of peptides with antihypertensive activity in functional food industry.

Purification and characterization of angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysate of hen egg white lysozyme

The aims of this study were to explore new functional bioactivity of hen egg white lysozyme (HEWL) protein hydrolysate and to identify potential angiotensin I-converting enzyme (ACE) inhibitory peptides from HEWL. The protein was hydrolyzed by gastrointestinal enzymes, including pepsin, α-chymotrypsin and trypsin, under simulated physiological conditions, and the hydrolysate exhibited potent ACE inhibitory activity with an IC50 value of 15.6±1.4μg/mL. Three novel ACE inhibitory peptides, specifically, Met-Lys-Arg, Arg-Gly-Tyr and Val-Ala-Trp with IC50 values of 25.7±0.2, 61.9±0.1 and 2.86±0.08μM, respectively, were isolated from the hydrolysate of HEWL in two stages of reverse-phase high-performance liquid chromatography (RP-HPLC), and their sequences were analyzed by UPLC–MS/MS. Kinetics studies suggested that the purified peptides were all competitive inhibitors; preincubation experiments implied that the peptide Arg-Gly-Tyr was a true substrate and that the other two peptides were true ACE inhibitors. These results indicate that HEWL may become an effective source of ACE inhibitory peptides. This study reveals the antihypertensive bioactivity of HEWL protein hydrolysate and provides further evidence that eggs are an excellent source of health-promoting food.

Angiotensin-I-converting enzyme (ACE)-inhibitory and antihypertensive properties of squid skin gelatin hydrolysates

Gelatin extracted from squid (Dosidicus eschrichitii Steenstrup) skin was hydrolysed with pepsin to prepare Angiotensin-I-Converting Enzyme (ACE) inhibitory peptide. The ACE-inhibitory activity was measured by spectrophotometric assay. The hydrolysate was fractionated into three ranges of molecular weight (6kDa<HSSG-I<10kDa, 2kDa<HSSG-II<6kDa, HSSG-III<2kDa) using an ultrafiltration unit. The HSSG-III showed the most potent ACE inhibitory activity in vitro with IC50 of 0.33mg/ml. Renovascular hypertensive rats (RHR) model was made with two-kidney one clip assay, and antihypertensive effects were studied in RHR treated with HSSG-III for 30days by oral administration. Arterial blood pressure were measured respectively. The HSSG-III remarkably reduced the arterial blood pressure of RHR. These results suggested that hydrolysate of squid skin gelatin obtained by treatment with pepsin was a good source of peptides with ACE-inhibitory activity and had an antihypertensive effect by oral administration.

Optimization of peptic hydrolysis parameters for the production of angiotensin I‐converting enzyme inhibitory hydrolysate from Acetes chinensis through Plackett–Burman and response surface methodological approaches

Angiotensin I-converting enzyme (ACE) plays an important physiological role in regulating blood pressure. The elevation of blood pressure could be suppressed by inhibiting ACE. ACE inhibitory peptides derived from food proteins could exert antihypertensive effects without side effects. Acetes chinensis is a marine shrimp suitable for the production of ACE inhibitory peptides. The principal objective of this study was to screen for the significant variables, and further to optimize the levels of the selected variables, for the enzymatic production of ACE inhibitory peptides from Acetes chinensis. Plackett-Burman design and response surface methodology were employed to optimize the peptic hydrolysis parameters of Acetes chinensis to obtain a hydrolysate with potent ACE inhibitory activity. The peptic hydrolysis variables were subject to a Plackett-Burman design for screening the main factors. The selected significant parameters such as pH, hydrolysis temperature and enzyme/substrate (E/S) ratio were further optimized using a central composite design. The optimized conditions were: pH 2.5, hydrolysis temperature 45 °C, E/S ratio 17 800 U kg(-1) shrimp and substrate concentration 200 g L(-1). The results showed that 3-5 h hydrolysis could result in a hydrolysate with ACE inhibition IC(50) of 1.17 mg mL(-1) and a high DH of 25-27%. Plackett-Burman design and RSM performed well in the optimization of peptic hydrolysis parameters of Acetes chinensis to produce hydrolysate with ACE inhibitory activity. A hydrolysate with potent ACE inhibitory activity and high degree of hydrolysis was obtained, so that the yield of ACE inhibitory peptides in it was high.

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 L 16 (4 5) 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 ( IC 50 = 100–200 μg/ml and 70 μg/ml, respectively), accompanied by high yield, high DH and strong bitterness. Furthermore, ACE inhibition was positively correlated ( r 2 = 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.