Enzyme Inhibitory Peptides Research Articles

Isolation, Evaluation, and Identification of Angiotensin I-Converting Enzyme Inhibitory Peptides from Game Meat.

Game meat has been underutilized, while it offers the potential to diversify not only the human diet but also increase food production and the nutritional value of meat products. This study aimed to determine the angiotensin I-converting enzyme (ACE) inhibitory activities of the digested game meats (venison and boar meat) compared with those of livestock meats (beef and pork). Through the sodium dodecyl sulfate polyacrylamide gel electrophoresis and size chromatography results, we found that the digested products from each meat had different molecular weights. The ACE inhibitory ratio in all tested samples had gradually increased following by the enzyme treatments. ACE inhibitory ratios and the half maximal inhibitory concentration values indicated that digested venison was the most potent inhibitor of ACE activity, followed by the digested boar meat. The level of anserine in digested venison was higher than that in the other meats, but the carnosine level was lower. Through fractionations and liquid chromatography–tandem mass spectrometry analysis, five ACE inhibitory peptides were identified from the digested venison. Of these peptides, Isoleucine-Lysine- Glutamic Acid-Valine-Threonine-Glutamic Acid-Arginine (IKEVTER) demonstrated the highest ACE inhibitory activity. Therefore, the game meat is food that is believed potentially to offer high bioactivities, particularly antihypertensive forces.

Biologically active peptides of meat and meat product proteins: a review. Part 2. Functionality of meat bioactive peptides

Biologically active peptides (BAP) are regarded as the main products of protein hydrolysis. BAP activity depends on the amino acid sequence molecular weight and chain length, type and charge of an amino acid at the N-terminus and C-terminus, hydrophobic and hydrophilic properties, spatial structure. They positively influence many systems of the human body, including the blood circulatory, nervous, immune, gastrointestinal and other systems. The health-improving effect of bioactive peptides is formed due to their antioxidant, antihypertensive, antithrombotic, immunomodulatory, antimicrobial, anti-allergic, opioid, anti-inflammatory, hypocholesterolemic and anticancer properties. Angiotensin-I-converting enzyme (ACE) inhibitory peptides are most studied due to their effect on blood pressure regulation. Unlike synthetic preparations, biologically active peptides do not have side effects and, therefore, can be used as their alternative. There is a growing commercial interest in peptides generated from meat proteins is in the context of health saving functional foods. The paper describes prospects, pros and cons of using bioactive peptides as functional food ingredients and biologically active food additives.

One peptide derived from hen ovotransferrin as pro-drug to inhibit angiotensin converting enzyme

Angiotensin I-converting enzyme (ACE) inhibitory peptide was derived from hen ovotransferrin and identified as Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr. It produced a concentration-dependent inhibition of ACE activity in vitro with an IC50 value of 102.8 μM. After hydrolysis by ACE, the product (Lys-Val-Arg-Glu-Gly-Thr) has an IC50 value of 9.1 μM that was about 11-fold lower of the parent peptide. Thus, Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr can be considered as a pro-drug. Moreover, Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr and Lys-Val-Arg-Glu-Gly-Thr were intravenously administered into spontaneously hypertensive rats (SHR) to monitor the time-course change of systolic blood pressure. We found that Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr and its hydrolyzed product produced the maximal reduction of systolic blood pressure at 40 min and 20 min after injection, respectively. This 20 min delay might be considered as the time required for conversion of prodrug into Lys-Val-Arg-Glu-Gly-Thr, the true ACE inhibitor. In conclusion, the obtained results suggest that Lys-Val-Arg-Glu-Gly-Thr-Thr-Tyr works as the pro-drug of Lys-Val-Arg-Glu-Gly-Thr to inhibit ACE activity in vivo.

Physicochemical Properties and Angiotensin I Converting Enzyme Inhibitory Peptides of Freshwater Fish Skin Collagens

ABSTRACT The objectives of this study were to characterize physicochemical properties of two collagens, tilapia skin (TS) and hybrid catfish skin (HS). Angiotensin-converting enzyme (ACE) inhibitory peptides from extracted TS and HS collagen using pepsin were also determined. HS collagen had a higher amount of imino acid than TS collagen, while TS contained higher amounts of tyrosine (Tyr) and lysine (Lys). Fourier-transform infrared spectra of both collagens showed predominant helix structure. The HS collagen hydrolysate prepared by pepsin was fractionated using sequential ultrafiltration membranes, and the fraction with molecular weight (MW) <5 kDa showed the highest ACE inhibitory activity (p < .05). After cation exchange and two steps size exclusion chromatography, peptides showed ACE inhibitory activity of 72.06%. This study revealed that ACE inhibitory peptides derived from HS collagen could be developed as a functional food with potential antihypertensive properties.

PURIFICATION AND CHARACTERISATION OF ANGIOTENSIN I CONVERTING ENZYME (ACE) INHIBITORY PEPTIDE FROM BLOOD COCKLE (Anadara granosa) MEAT HYDROLYSATE

Blood cockle (Anadara granosa) is the most abundant and available bivalves in Malaysia. Blood cockles meat has high protein content and has potential to generate bioactive peptides. To date, no study has been reported on purification and identification of angiotensin I converting enzyme (ACE) inhibitory peptides from blood cockle meat. Thus, the objectives of this study were to purify and characterize ACE inhibitory peptide from blood cockle meat hydrolysate. ACE inhibitory peptides from blood cockle meat hydrolysate (CMH) were prepared by enzymatic protein hydrolysis using Protamex®. Crude CMH was characterized for its stability against gastrointestinal proteases, at varying pH (2–11) and temperature (4–90°C). Next, crude CMH was purified by ultrafiltration, ion exchange chromatography and reverse-phase chromatography and its amino acid sequence was identified. It was found that crude CMH was highly stable at low pH and temperature, and was resistant to gastrointestinal proteases (pepsin and trypsin). A three-step purification increased the inhibitory activity of CMH, reducing its IC50 from 0.35 mg/ml to 0.0094 mg/ml. The amino acid sequence of the purified peptide was determined as VNDLLSGSFKHFLY, with a molecular weight of 1621.88 Da. This study suggested the potential of ACE inhibitory peptide derived from cockle meat as a nutraceutical ingredient in functional food

Angiotensin-I converting enzyme inhibitory peptide derived from the shiitake mushroom (Lentinula edodes).

Angiotensin-I converting enzyme (ACE) inhibitors are widely used to control hypertension. In this study, protein hydrolysates from shiitake mushroom were hydrolyzed to prepare ACE-inhibitory peptides. Optimum process conditions for the hydrolysis of shiitake mushrooms using Alcalase were optimized using response surface methodology. Monitoring was conducted to check the degree of hydrolysis (DH) and ACE inhibitory activity. In the results, the optimum condition with the highest DH value of 28.88% was 50.2°C, 3-h hydrolysis time, and 1.16 enzyme/substrate ratios. The highest ACE inhibitory activity (IC50 of 0.33μg/mL) was under 47°C, 3h 28min hydrolysis time, and 0.59 enzyme/substrate ratios. The highest activity was fractionated into 5 ranges of molecular weight, and the fraction below 0.65kDa showed the highest activity with IC50 of 0.23μg/mL. This fraction underwent purification using RP-HPLC, meanwhile the peak which offered a retention time of about 37min showed high ACE inhibitory activity. Mass spectrometry identified the amino acid sequence of this peak as Lys-Ile-Gly-Ser-Arg-Ser-Arg-Phe-Asp-Val-Thr (KIGSRSRFDVT), with a molecular weight of 1265.43Da. The synthesized variant of this peptide produced an ACE inhibitory activity (IC50) of 37.14μM. The peptide KIGSRSRFDVT was shown to serve as a non-competitive inhibitor according to the Lineweaver-Burk plot findings. A molecular docking study was performed, which showed that the peptide binding occurred at an ACE non-active site. The findings suggest that peptides derived from shiitake mushrooms could serve either as useful components in pharmaceutical products, or in functional foods for the purpose of treating hypertension.

Effect of the Casein-Derived Peptide Met-Lys-Pro on Cognitive Function in Community-Dwelling Adults Without Dementia: A Randomized, Double-Blind, Placebo-Controlled Trial.

BackgroundPreventative measures have recently been taken to reduce the incidence of Alzheimer’s disease worldwide. We previously showed that Met-Lys-Pro (MKP), a casein-derived angiotensin-converting enzyme inhibitory peptide with the potential to cross the blood–brain barrier, attenuated cognitive decline in a mouse model of Alzheimer’s disease. However, the effect of MKP on cognitive function improvement in humans remains unknown. This exploratory study sought to investigate whether MKP intake could improve cognitive function in adults without dementia.MethodsA total of 268 community-dwelling adults without dementia participated in this 24-week randomized controlled trial. Participants were randomly allocated to the MKP (n = 134) or placebo (n = 134) group. The MKP group received four tablets daily, each containing 50 μg MKP, while the placebo group received four dextrin tablets containing no detectable MKP for 24 weeks. Scores on the Japanese version of the cognitive subscale of the Alzheimer’s Disease Assessment Scale (ADAS-cog) were used as the primary outcome to compare cognitive function between the MKP and placebo groups. The study products were also evaluated for safety.ResultsThe intention-to-treat analysis showed that there was no significant difference between the groups in terms of the ADAS-cog total score. Orientation, as measured by the respective ADAS-cog subscale, was significantly improved compared to placebo at 24 weeks post-MKP administration (P = 0.022). No serious adverse events due to MKP intake were observed.ConclusionTo the best of our knowledge, this is the first study to report the effects of MKP on human cognition. These preliminary results suggested the safety of daily MKP intake and its potential to improve orientation in adults without dementia. Further clinical studies are needed to confirm the present findings and the benefits of MKP on cognitive function.

Bioactive peptides derived from human milk proteins: an update.

Human milk contains a variety of bioactive proteins, and some of the bioactivities are exerted only after proteins are digested in the gastrointestinal tract. This review aims to overview recent studies on bioactive peptides in human milk and gastric digesta of breast-fed infants. Milk protein-derived peptides are endogenously present in human milk, and some of them have been reported to be bioactive peptides, such as a homologue of caseinophosphopeptide, an antimicrobial peptide, and an immunomodulatory peptide. A larger number of peptides are identified in gastric aspirates from breast-fed infants, and bioactive peptides such as angiotensin I-converting enzyme-inhibitory peptides, an antioxidative peptide, opioid agonist peptides are only found in the digesta of human milk but not in human milk per se. Casein is the greatest source of released bioactive peptides. Technological advances have considerably increased our capability to identify and characterize peptides derived from human milk proteins. However, their physiological significance and the roles of these bioactive peptides in growth and development of breast-fed infants have not yet been sufficiently elucidated, and further in-vivo experiments and clinical trials are warranted.

Identification of novel angiotensin I‐converting enzyme inhibitory peptide from collagen hydrolysates and its molecular inhibitory mechanism

SummaryThe fish collagen protein was hydrolysed and further fractionated into four molecular weight ranges by ultrafiltration. Subsequently, the peptide fraction with the potent angiotensin I‐converting enzyme (ACE) inhibitory activity was identified. The potential inhibitory mechanism of the peptide was clarified by molecular docking. As a result, FCPH‐Ⅳ with molecular weight between 600 and 1000 Da exerted the high ACE inhibitory activity and was identified by de novo peptide sequencing. The peptide GHVGAAGS exhibited significant ACE inhibitory activity with the IC50 value of 407.28 ± 3.55 μm. In addition, the docking results showed the interactions between the amino acids at the four positions closest to the C‐terminal site of GHVGAAGS and the major active residues (GLN281, HIS353, LYS511 and HIS513) of ACE lead to the conformational change in ACE. This work indicates that fish collagen could be utilised to produce ACE inhibitory peptides and develop health products.

Characterizing the Binding of Angiotensin Converting Enzyme I Inhibitory Peptide to Human Hemoglobin: Influence of Electromagnetic Fields.

Drug-protein complexes is one of the crucial factors when analyzing the pharmacokinetics and pharmacodynamics of a drug because they can affect the excretion, distribution, metabolism and interaction with target tissues. The aim of this study was to investigate the interaction of human hemoglobin (Hb) and angiotensin I converting enzyme inhibitory peptide (ACEIP) in the absence and presence of different- frequency electromagnetic fields (EMF). Various spectroscopic methods like fluorescence spectroscopy, ultraviolet, circular dichroism and conductometry techniques were applied to investigate Hb-ACEIP interaction in the absence and presence of EMF. The presented spectroscopic studies indicated that EMF changed the interaction between Hb and ACEIP. The a-helix content of Hb decreased upon binding to ACEIP and conductivity of the solution enhanced upon binding. Based on Stern-Volmer equations, it could be stated that the Hb-ACEIP affinity was higher in the presence of EMF. It can be concluded that for patients who use the drug to control blood pressure, a low-frequency electromagnetic field would have a positive effect on the uptake of the drug.

Identification and molecular mechanism of angiotensin-converting enzyme inhibitory peptides from Larimichthys crocea titin

This study aimed to identify novel ACEI peptides from Larimichthys crocea titin using in silico approaches and to clarify the molecular interaction mechanism. The hydrolyzed peptides of titin were compared with known ACEI peptides in the AHTPDB and BIOPEP-UWM database. Furthermore, peptides were evaluated for their solubility, ADMET properties, △G (kcal/mol) values, and in vitro ACEI activity. Molecular mechanism of ACE-peptide was performed by molecular interactions and binding orientation study. The results revealed that IC50 values of Trp-Ala-Arg(WAR) and Trp-Gln-Arg(WQR) were (31.2 ± 0.8) and (231.33 ± 0.02) μmol/L, respectively. The docking interactions result suggested that ACE-WAR and ACE-WQR complexes have same binding site, including the residues LYS511, TYR520, TYR523, HIS353, and HIS513. Molecular docking of two tripeptides WAR and WQR with ACE studies predicted their binding site and clarified the interaction between ACE and its inhibitors. The molecular docking data are consistent with the ACE inhibitory activity of the studied peptides. The results showed that Larimichthys crocea titin may be a valuable source for developing nutraceutical food.

Angiotensin I-converting Enzyme (ACE) Inhibitory Activity of Ace Inhibitory Peptides Produced during the Fermentation of Pigeon Pea (Cajanus cajan) Tempe

Fermentation products are common sources of angiotensin I-converting enzyme (ACE) inhibitory peptides used for hypertension treatment. This research investigated the effect of fermentation time on the ACE inhibitory activity produced during the fermentation of pigeon pea tempe and aimed to determine the optimal fermentation time to obtain pigeon pea tempe with the highest ACE inhibitory activity. Seeds were inoculated with Raprima® (0.02% w/w) containing Rhizopus oligosporus spores and fermented for 0-96 h. Protein pattern, degree of hydrolysis (DH), soluble protein content and ACE inhibitory activity were observed during fermentation. The result from SDS-PAGE shows that protein hydrolysis occurred after 12 h fermentation, marked by the appearance and greater intensity of protein bands with low-molecular-weight (60 kDa). An increase in DH and soluble protein content were detected during the fermentation and reached a maximum of 23.99% and 3.15 mg mL -1 at 96 h fermentation, respectively. The ACE inhibitory activity increased with fermentation time and pigeon pea tempe fermented for 48 h (76.14%) has the highest ACE inhibitory activity with IC 50 values of 0.65 mg mL -1 . It could be concluded that the optimal fermentation time to obtained pigeon pea tempe with the highest ACE inhibitory activity is for 48 h of fermentation.

Purification and the secondary structure of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from the alcalase hydrolysate of seahorse protein.

Bioactive peptides with blood pressure-lowering functions have received increasing attention. In recent years, many ACE-inhibiting peptides have been widely purified from various food-derived proteins and have received considerable interest owing to their potential role in cardiovascular diseases and in the reduction of side effects. In this study, we hydrolyzed a three-spot seahorse (Hippocampus trimaculatus Leach) protein by alcalase to obtain a hydrolysate containing angiotensin I-converting enzyme (ACE) inhibitory peptide. Then, the hydrolysate was fractionated by dialysis, Sephadex G-25 gel filtration chromatography, and reverse-phase high performance liquid chromatography. After consecutive purification, a potent ACE-inhibiting peptide composed of 8 amino acids (Pro-Ala-Gly-Pro-Arg-Gly-Pro-Ala; MW: 721.39Da; IC50 value: 7.90μM) was successfully isolated from three-spot seahorse protein. For the first time, a novel ACE-inhibiting peptide (PAGPRGPA) was isolated from the seahorse. Circular dichroism (CD) analyses suggested that the secondary structure of the purified peptide was mainly composed of random coil. Therefore, the peptide from seahorse protein may be used as a favorable ingredient in nutraceuticals, medicines, and functional foods against antihypertensive and related diseases.

Successive digestion of tilapia collagen by serine proteinase and proline specific endopeptidase to produce novel angiotensin I-converting enzyme inhibitory peptides

Serine proteinase, purified from the hepatopancreas of Pacific white shrimp (Litopenaeus vannamei), was used to hydrolyze acid solubilized collagen (ASC) isolated from Nile tilapia (Oreochromis sp.) skin to produce angiotensin I-converting enzyme (ACE) inhibitory peptides (ACEIPs). A series of column chromatography assays were used to separate the ACEIPs. A peptide, NPARTCR, was isolated as it exhibited high ACE inhibition potential. Further digestion of this peptide by a proline specific endopeptidase (PSEP), produced a pentapeptide ARTCR with ACE inhibitory activity (IC50) of 77.0 μmol/L. Both NPARTCR and ARTCR inhibited ACE in a non-competitive manner. An in vivo study in rats demonstrated that ARTCR has ACE inhibitory activity via lowering systolic blood pressure in spontaneously hypertensive rats (SHRs). These results suggest that processing by-products from shrimp and tilapia are ideal raw materials for the production of serine proteinase and collagen, respectively. Serine proteinase and collagen are both ideal raw materials that can be used to derive ACE inhibitory active peptides against hypertension.

An efficient controlled release strategy for hypertension therapy: Folate-mediated lipid nanoparticles for oral peptide delivery

Hypertension is an important cardiovascular disease, which need long-term medication. Thus, oral drug delivery system is a preferred route for hypertension patients due to the convenience and compliance. Val-Leu-Pro-Val-Pro (VLPVP, VP5) is an angiotensin converting enzyme inhibitory peptide with antihypertensive effects. However, the oral peptide delivery is faced with obstacles, such as gastric acid, enzyme degradation and intestine barriers. Herein, we developed a controlled release system consisting of a PLGA core encapsulated with VP5 and a folate-decorated lipid shell (FA-VP5-LNPs) for the oral delivery of antihypertensive peptide. The results found that FA-VP5-LNPs exhibited high stability and possessed a controlled release behavior. Besides, FA-VP5-LNPs improved the cellular uptake both in Caco-2 and HT29 cells and enhanced in situ intestinal absorption in SD rats. The in vivo bioavailability study showed a superior oral absorption of FA-VP5-LNPs, and the AUC0-72 h of FA-VP5-LNPs was 30.71-fold higher than that of free VP5. The pharmacodynamics study exhibited that FA-VP5-LNPs maintained strong antihypertensive effect for six days compared with free VP5, which may reduce the frequency of administration and improve patient compliance. In addition, the nano-formulations showed no toxicity to cells and tissues. These promising results suggested that FA-VP5-LNPs could overcome the intestinal barrier and provide a potential strategy for enhancing peptide delivery and improve the antihypertensive effects.

Purification and characterization of angiotensin-I-converting enzyme inhibitory peptides isolated from whey proteins of milk fermented with Lactobacillus plantarum QS670

An angiotensin-converting enzyme inhibitory (ACEI) peptide with a median inhibitory concentration (IC50) of 1.26 mg/mL was purified from whey proteins resulting from a fermentation using Lactobacillus plantarum QS670. The peptide was subsequently derived from an αS1-casein, κ-casein, β-lactoglobulin, or serum albumin fraction. Analysis via liquid chromatography tandem mass spectrometry indicated that it had an amino acid sequence of Gly-Ala (GA). The GA dipeptide was also synthesized using an Fmoc solid-phase method. The GA dipeptide exhibited an IC50 of 1.22 mg/mL and was shown to be stable across both temperature (20 to 60°C) and pH (2 to 12). Digestive enzymes including pepsin, trypsin, and chymotrypsin had negligible effects on activity. The whey exerted hypotensive effects when fed to spontaneously hypertensive rats (SHR), which exhibited a blood pressure drop of 2.33 kPa. A 4-wk gavage treatment resulted in greater decreases of 7.46 kPa. Results of this study indicate that milk fermented using Lb. plantarum QS306 has potential to be used as a functional food to help prevent or reduce hypertension-associated diseases.

Microbiological characterization of Gioddu, an Italian fermented milk

Gioddu, also known as “Miciuratu”, “Mezzoraddu” or “Latte ischidu” (literally meaning acidulous milk), is the sole variety of traditional Italian fermented milk. The aim of the present study was to elucidate the microbiota and the mycobiota occurring in artisan Gioddu sampled from three Sardinian producers by combining the results of viable counting on selective culture media and high-throughput sequencing. Physico-chemical parameters were also measured. The overall low pH values (3.80–4.22) recorded in the analyzed Gioddu samples attested the strong acidifying activity carried out by lactic acid bacteria during fermentation. Viable counts revealed the presence of presumptive lactococci, presumptive lactobacilli and non-Saccharomyces yeasts. A complex (kefir-like) microbiota of bacteria and yeasts was unveiled through sequencing. In more detail, Lactobacillus delbrueckii was found to dominate in Gioddu together with Streptococcus thermophilus, thus suggesting the establishment of a yogurt-like protocooperation. Unexpectedly, in all the three analyzed batches from two out of the three producers Lactobacillus kefiri was also detected, thus representing an absolute novelty, which suggests the presence of bioactive compounds (e.g. exopolysaccharides) similar to those characterizing milk kefir beverage. Mycobiota population, studied for the very first time in Gioddu, revealed a more complex composition, with Kluyveromyces marxianus, Galactomyces candidum and Geotrichum galactomyces constituting the core species. Further research is needed to disclose the eventual occurence in Gioddu of probiotic cultures and bioactive compounds (e.g. exopolysaccharides, angiotensin-converting enzyme inhibitory peptides and antimicrobial compounds) with potential health-benefits for the consumers.

Purification, Characterization and Mechanistic Evaluation of Angiotensin Converting Enzyme Inhibitory Peptides Derived from Zizyphus Jujuba Fruit

The synthetic Angiotensin Converting Enzyme (ACE) inhibitors have side effects and hence demands for natural ACE inhibitors have been rising. The aim of this study is to purify and introduce natural ACE inhibitors extracted from Zizyphus jujuba fruits. Proteins from Zizyphus jujuba were lysed by trypsin, papain and their combination. Acquired peptides were purified and evaluated for ACE inhibitory activity. Peptide fractions with inhibitory activity were sequenced using tandem mass spectrometry. To elucidate the mode of peptide binding to ACE, homology modeling, molecular docking and molecular dynamics simulation were performed. Amino acid sequence of F2 and F4 peptides, which were the most active hydrolysates, were determined to be IER and IGK with the IC50 values of 0.060 and 0.072 mg/ml, respectively. Results obtained by computational analysis revealed that similar to the common ACE competitive inhibitors such as captopril, IER tripeptide binds to the enzyme active site, in vicinity of the zinc binding site, and occupies the S1 and S2’ subsites. Binding occurs through hydrogen bonding with Gln293, Lys522, His524, Tyr531 and also several hydrophobic interactions. Collectively, these findings indicate that IER tripeptide inhibits the rabbit ACE enzyme through a competitive mechanism of inhibition with IC50 values in the millimolar range.

Preparation of ACE-inhibitory peptides from milk protein in continuous enzyme membrane reactor with gradient dilution feeding substrate

In this study, a novel method of gradient dilution feeding substrate (GDFS) was established to improve the yield of angiotensin-converting enzyme (ACE) inhibitory peptides from milk protein. The hydrolysis process stability, enzymatic efficiency and kinetics of the method were studied and compared with traditional feeding modes, viz., adding water after feeding substrate or constant concentration feeding substrate. Results showed that the GDFS mode achieved the highest membrane flux and lowest fluctuation of protein concentration in the reactor. Moreover, the GDFS maximized protein conversion rate, yield of peptides, and ACE-inhibitory activity, with their values of 67.58 %, 138.51 g/(g*Neutrase), and 0.74 mg/mL (IC50), respectively. In further study, the kinetic model of GDFS mode was successfully established with KM of 69.481 g/L and Vmax of 0.752 g·L−1 min−1. Based on the optimum condition of the kinetic model, the practical longest runtime was 720 min. Obtained results suggested that GDFS mode could be used as an alternative method in the preparation of high-yield bioactive peptides.

Immobilized metal affinity chromatography matrix modified by poly (ethylene glycol) methyl ether for purification of angiotensin I-converting enzyme inhibitory peptide from casein hydrolysate

Purification of small bioactive peptides from complex biological samples is a difficult task due to the interference of concentrated large biomolecules. In this study, a magnetic immobilized metal affinity chromatography matrix modified by poly (ethylene glycol) methyl ether (IMACM@mPEG) was prepared and applied for the rapid purification of angiotensin I-converting enzyme (ACE) inhibitory peptides from casein hydrolysate. The proposed IMACM@mPEG considerably reduced the non-specific adsorption of large proteins and exhibited improved purification efficiency towards ACE inhibitory peptides. A novel peptide with moderate ACE inhibitory activity (IC50 value of 274 ± 5 μM) was identified as LLYQEPVLGPVR. Lineweaver-Burk plot confirmed the non-competitive inhibition pattern of LLYQEPVLGPVR. The purified peptide was digested after simulated gastrointestinal digestion and produced shorter peptides which contributed to enhanced ACE inhibitory activity. These results indicated that the IMACM@mPEG is an effective method for the prepurification of ACE inhibitory peptide and the purified peptide LLYQEPVLGPVR may have potential as nutraceutical ingredient in functional foods for hypertension treatments.