Inhibition of angiotensin I-converting enzyme by wheat gliadin hydrolysates

A tryptic gliadin hydrolysate was fractionated into peptide fractions, which were assigned to either the central domain (CD) or terminal domains (TD) of gliadins. The domains were expected to contain amino acid (AA) sequences which, when released from the parent protein, inhibit the angiotensin I-converting enzyme (ACE), which plays a key role in regulating blood pressure. A proline (Pro) poor TD related fraction, containing the smallest peptides, showed the highest ACE inhibitory activity (IC₅₀ = 0.33 mg/ml). Additional peptidases were selected based on their in silico predicted ability to release ACE inhibitory peptides. Further hydrolysis of the tryptic hydrolysate fractions with thermolysin, Clarex, Alcalase and Esperase increased ACE inhibitory activities. Immobilised Ni²⁺-ion affinity chromatography (IMAC) purification of a TD related peptide fraction obtained by sequential hydrolysis with trypsin and thermolysin yielded a fraction with an IC₅₀ value of 0.02 mg/ml. This IMAC fraction was enriched in histidine and hydrophobic AA (Pro, Val, Ile, Leu and Phe).     

Vascular effects of egg white‐derived peptides in resistance arteries from rats. Structure–activity relationships

BACKGROUND: The vasodilator properties of several peptide sequences derived from egg white proteins were screened in mesenteric resistance arteries from Wistar‐Kyoto rats. For this, third‐order branches of the mesenteric arteries from 6‐month‐old male rats were used. The vasodilator responses, with or without endothelium, to several peptides (0.1 mmol L^?1) were analysed in an isometric myograph. Moreover, the effect of nitric oxide (NO) synthase (L‐NAME, 100 µmol L^?1) and cyclooxygenase (indomethacin, 10 µmol L^?1) inhibitors on the vasodilator response was tested. RESULTS: The peptides Arg‐Ala‐Asp‐His‐Pro‐Phe‐Leu, Arg‐Ala‐Asp‐His‐Pro‐Phe, Arg‐Ala‐Asp‐His‐Pro, Tyr‐Arg‐Gly‐Gly‐Leu‐Glu‐Pro‐Ile‐Asn‐Phe, Arg‐Asp‐Ile‐Leu‐Asn‐Gln and Val‐Pro‐Pro showed a high endothelium‐dependent vasorelaxation, whereas Phe‐Arg‐Ala‐Asp‐His‐Pro‐Phe‐Leu was only partially endothelium‐dependent. The relaxation induced by Arg‐Ala‐Asp‐His‐Pro‐Phe‐Leu, Arg‐Ala‐Asp‐His‐Pro‐Phe, Arg‐Ala‐Asp‐His‐Pro, Arg‐Asp‐Ile‐Leu‐Asn‐Gln and Val‐Pro‐Pro was mainly mediated by NO, since the response was inhibited only by L‐NAME, while both L‐NAME and indomethacin inhibited the vasodilator response induced by Phe‐Arg‐Ala‐Asp‐His‐Pro‐Phe‐Leu and Tyr‐Arg‐Gly‐Gly‐Leu‐Glu‐Pro‐Ile‐Asn‐Phe. The presence of Arg or Tyr at the N‐terminal position could be related to the vasodilator activity of these compounds in this vascular bed. The well‐known angiotensin‐converting enzyme inhibitor captopril showed only a slight vasodilator effect. CONCLUSION: These peptides could reduce the vascular resistance and be used as functional ingredients in the prevention and/or treatment of hypertension and other associated disorders. Copyright © 2010 Society of Chemical Industry     

Antioxidant activity of yoghurt peptides: Part 2 – Characterisation of peptide fractions

The aim of the present study was to elucidate previous findings showing that peptide fractions isolated from yoghurt had antioxidant effects. Therefore, peptides and free amino acids released during fermentation of milk were characterised. Yoghurt samples were stripped from sugars and lactic acid and subsequently fractionated by ultra filtration using membranes with cut off sizes of 30, 10 and 3 kDa. The peptides in these fractions were identified by LC–MS/MS. The identified peptides comprised a few N-terminal fragments of αs₁-, αs₂-, and κ-casein, and several fragments from β-casein. Almost all the peptides identified contained at least one proline residue. Some of the identified peptides included the hydrophobic amino acid residues Val or Leu at the N-terminus and Pro, His or Tyr in the amino acid sequence, which is characteristic of antioxidant peptides. In addition, the yoghurt contained a considerable amount of free amino acids such as His, Tyr, Thr and Lys, which have been reported to have antioxidant properties. Thus, our findings confirm that the antioxidant effects of the peptide fractions from yoghurt are due to the presence of certain peptides and free amino acids with recognised antioxidant activity in these fractions.     

ANGIOTENSIN I-CONVERTING ENZYME (ACE) INHIBITORY PEPTIDES FROM WHEY FERMENTED BY LACTOBACILLUS SPECIES

From 2% (w/w) whey powder in growth media, inhibitory peptides against angiotensin I-converting enzyme (ACE) were studied with nine Lactobacillus species. Lb. brevis, Lb. helveticus and Lb. paracasei were proved to be the most effective strains in liberating ACE inhibitory peptides from whey protein. The inhibition rates of these peptides against ACE ranging from 93.3 to 100%. Several distinct peaks were eluted when the whey proteins were fractionated on a Delta Pak C₁₈ column by reversed phase-high performance liquid chromatography (RP-HPLC). Among ACE inhibitory activities of 14 peptides purified by dialysis and by fractionation using RP-HPLC, two peptide fractions (H5 and H7) of Lb. helveticus showing IC₅₀ values of 5.3 and 7.8 were the most potent ACE inhibitors.
All of these peptides including some other peptides (H1 and B1), having strong inhibitory activities against ACE were pentapeptides positioning with Ala at their N-terminal and these petapeptides had mostly hydrophobic (Pro, Val and Leu) or aromatic (Phe) amino acids at the C-terminal.

PRACTICAL APPLICATIONS

There is a significant amount of research and interest in developing and charactering the peptides that inhibit angiotensin I-converting enzyme (ACE) activity as these natural products may have a role in blood pressure control in man. This study revealed that the identification of peptides, mostly composed of pentapeptides following fermentation of whey protein in growth medium with different strains have the ACE inhibitory activities. These peptides may have antihypertensive effect as natural and safe nutraceutical/functional ingredients, though the exact potency of the pentapeptides isolated in this experiment has not been determined.     

Two Novel Bioactive Peptides from Antarctic Krill with Dual Angiotensin Converting Enzyme and Dipeptidyl Peptidase IV Inhibitory Activities

Inhibition of dipeptidyl peptidase IV (DPP‐IV) and angiotensin converting enzyme (ACE) are considered useful in managing 2 often associated conditions: diabetes and hypertension. In this study, corolase PP was used to hydrolyze Antarctic krill protein. The hydrolysate (AKH) was isolated by ultrafiltration and purified by size‐exclusion chromatography, ion exchange chromatography and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) sequentially. The in vitro inhibitory activities of all AKHs and several fractions obtained against ACE and DPP‐IV were assessed. Two peptides, purified with dual‐strength inhibitory activity against ACE and DPP‐IV, were identified by TOF‐MS/MS. Results indicated that not all fractions exhibited dual inhibitory activities of ACE and DPP‐IV. The purified peptide Lys‐Val‐Glu‐Pro‐Leu‐Pro had half‐maximal inhibitory concentrations (IC₅₀) of 0.93±0.05 and 0.73±0.04 mg/mL against ACE and DPP‐IV, respectively. The other peptide Pro‐Ala‐Leu had IC₅₀ values of 0.64±0.05 and 0.88±0.03 mg/mL against ACE and DPP‐IV, respectively. This study firstly reported the sequences of dual bioactive peptides from Antarctic krill proteins, further provided new insights into the bioactive peptides responsible for the ACE and DPP‐IV inhibitory activities from the Antarctic krill protein hydrolysate to manage hypertension and diabetes.     

IDENTIFICATION OF ANGIOTENSIN I-CONVERTING ENZYME INHIBITORY PEPTIDES DERIVED FROM THE PEPTIC DIGEST OF SOYBEAN PROTEIN

Peptidic fractions which inhibit angiotensin I‐converting enzyme (ACE) were separated from peptic digests of soybean by ion exchange chromatography and gel filtration. Further separation of the peptidic fractions by ODS HPLC afforded active peptides, the amino add sequences of which were identified by Edman's procedure as: Ile‐Ata (inhibitory against ACE with an IC₅₀of 153 μM), Tyr‐Leu‐Ala‐Gly‐Asn‐Gln (14 μM), Phe‐Phe‐Leu (37 μM), Ile‐Tyr‐Leu‐Leu (42 μM), and Val‐Met‐Asp‐Lys‐Pro‐Gln‐Gly (39 μM). The antihypertensive activity of the soybean peptides was also investigated. Peptide fractions (2.0 g/kg body weight, oral administration) markedly towered the blood pressure of spontaneously hypertensive rats (SHRs).     

Purification and identification of novel antioxidative peptide released from Black-bone silky fowl (Gallus gallus domesticus Brisson)

Papain-treated Black-bone silky fowl (BSF) muscle hydrolysate was subjected to 6 kDa cutoff membrane ultrafiltration, and the resulting BSF peptides (<6 kDa) were purified by two-step reverse-phase high-performance liquid chromatography. The molecular weight (MW) distribution and amino acid composition were investigated for characterization of the BSF peptides. The results showed that the major amino acids of BSF peptides were Glu, Tyr, Lys, Asp, Leu, Ala, Thr and Pro, and the MW was from 281 to 7,982 Da. BSF peptides exhibited a strong antioxidant capacity. At 10 mg/mL, they displayed more powerful $ {\text{O}}_{2}^{ \cdot - } $ , DPPH^· and ABTS^·+ scavenging activity and reducing power than carnosine. The peptide fraction 8 with more hydrophilicity revealed stronger $ {\text{O}}_{2}^{ \cdot - } $ and ABTS^·+ scavenging activity and reducing power than BSF peptides and carnosine. Besides, a peptide, separated from fraction 8 and showed the strongest antioxidant capacity, was purified and identified by LC-ESI-MS/MS to be Glu-Pro-Asp-Arg-Tyr (678 Da).     

Evaluation of Antioxidant Activities of Zein Protein Fractions

Zein protein was extracted from the by‐product corn gluten meal. The obtained zein protein was 1st hydrolyzed by 4 different proteases. The antioxidant activities of the hydrolysates or peptides were evaluated by free radical scavenging activity, metal ion chelating activity, and lipid peroxidation inhibitory capacity. Among hydrolysates produced, alkaline protease hydrolysates exhibited the highest antioxidant activity. A regression model was established by uniform design to optimize the alkaline protease hydrolysis conditions. The hydrolysates with molecular weight < 3 kDa obtained from ultrafiltration showed the highest antioxidant activities in all relevant assays. The hydrolysates with molecular weight <3 kDa were subsequently purified by gel filtration chromatography, and fraction F3 exhibited the highest antioxidant activities. Two peptides were identified from fraction F3 using LC‐ESI‐Q‐TOF MS/MS as Pro‐Phe (263.13 Da) and Leu‐Pro‐Phe (375.46 Da). These peptides exhibited good free radical scavenging activity and lipid peroxidation inhibitory effect. The results clearly indicated that zein protein fractions are good sources for the development of natural antioxidants for the food industry.     

大豆生物活性肽的分离及其抗氧化活性研究

为制各具有抗氧化活性的大豆生物活性肽,本实验采用酶解处理大豆蛋白粉,对酶解产物进行葡聚糖Sephadex G-25凝胶柱层析分离,并对其各组分分子量分布及其抗氧化性进行了研究,对抗氧化活性最佳的肽段进行氨基酸组成分析。结果显示,大豆蛋白酶解物经Sephadex G-25分离后得到了六个肽片段,其中平均链长为4,即含有2～6个氨基酸残基的大豆功能短肽SPP4,对羟自由基具有最佳的清除作用,清除率达到80．13%,氨基酸组成分析发现该组分中缬氨酸、亮氨酸、苯丙氨酸、赖氨酸含量较高。结果表明,含2～6个氨基酸残基的大豆生理活性短肽具有较好的抗氧化活性。     

Angiotensin I-converting enzyme inhibitory activity of enzymatic hydrolysates of goat milk protein fractions

Casein and whey protein fractions from goat milk were hydrolysed by subtilisin and trypsin, individually and in combination, to release angiotensin converting enzyme (ACE)-inhibitory peptides. Selected hydrolysates were fractionated by size exclusion chromatography (SEC) and further characterised. The highest ACE-inhibitory activity was obtained from the casein fraction hydrolysed by the combination of enzymes. SEC presented 4 fractions with fraction F2 (<2.3 kDa) containing the highest concentration of peptides and the highest activity. F2 contained a number of peptides not previously identified from caprine caseins but with structural similarity to other ACE-inhibitory peptides. The most active fraction in relation to protein content was F4 with IC₅₀ between 9.3 and 5.1 μg mL⁻¹. This fraction contained a compound tentatively identified as WY, an active dipeptide not previously reported from caseins. The high inhibitory capacity of these fractions points towards the advantage of implementing a membrane process to concentrate the most active peptides.     

Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b5 reductase for the binding of β-NADH

Well-conserved three consecutive Pro residues (Pro247–249) in the NADH-binding subdomain of NADH-cytochrome b₅ reductase were proposed to form a basal part of the NADH-binding site. To investigate the structural and mechanistic roles of these residues, we expressed site-directed mutants for a soluble domain of the porcine enzyme where each of the residues was replaced with either Ala or Leu residue, respectively, using a heterologous expression system in Escherichia coli. Six mutants (P247A, P247L, P248A, P248L, P249A, and P249L) were produced as a fusion protein containing a 6×His-tag sequence at the NH₂-terminus and were purified to homogeneity with a stoichiometric amount of bound FAD. Mutations were each confirmed for the purified proteins by MALDI-TOF mass spectrometry. Steady-state kinetic analyses for NADH:ferricyanide reductase and NADH:cytochrome b₅ reductase acitivities were conducted for all the mutants. Substitution of Pro247 with Leu residue was found to significantly decrease k_cat with slight increase in K_m for the physiological electron donor NADH. However, K_m values for the electron acceptors (both cytochrome b₅ and ferricyanide) of P247L were found to be decreased significantly. Such changes were not observed for P247A or other four mutants. These results suggested that Pro247 among the three consecutive Pro residues has the most important role for the formation of a binding site cavity and that only a slight change in the side-chain volume at this residue from Ala to Leu residue affected the electron transfer reaction from NADH and, further, on the recognition of ferricytochrome b₅.     

Moisture‐Absorption and Water Dynamics in the Powder of Egg Albumen Peptide,Met‐Pro‐Asp‐Ala‐His‐Leu

Moisture absorbed into the powder of Met‐Pro‐Asp‐Ala‐His‐Leu (MPDAHL)—a novel egg albumen antioxidant peptide—profoundly affects its properties. In this study, we elucidated water dynamics in MPDAHL using DVS, DSC, and low‐field ¹H NMR. Based on the DVS data, we found that MPDAHL sorption kinetics obey a parallel exponential model. DSC results indicated that both water and heating could change the microstructure of MPDAHL. The T₂ parameters of NMR reflected the different phases of moisture absorption revealed that there were 4 categories of water with different states or mobility in the MPDAHL during the moisture absorption process. The fastest fraction T_2b mainly dominated the hygroscopicity of MPDAHL and the absorbed water significantly changed the proton distribution and structure of MPDAHL. Thus, this study shows that DVS, DSC, and low‐field ¹H NMR are effective methods for monitoring water mobility and distribution in synthetic peptides. It can be used to improve the quality assurance of functional peptides.     

Identification of active peptides from backbones of Nemipterus japonicus and Exocoetus volitans by electrospray ionisation–mass spectrometry

In our present investigation, Nemipterus japonicus and Exocoetus volitans backbone protein were hydrolysed by proteases like trypsin and pepsin, respectively. The protein hydrolysates were purified by different chromatographic methods, and the resulted purified peptides were analysed for their amino acid sequences by electrospray ionisation–MS/MS. The analysis of peptides showed sequences as Gly‐His‐Met‐Ser (451.8 Da) and Leu‐Glu‐Val‐Lys‐Pro (596.9 Da) for N. japonicus and E. volitans muscle, respectively. The presence of hydrophobic amino acids contributes more to the antioxidant activities of peptides than other amino acids. Moreover, sequence of amino acids in peptides plays an important role in their antioxidant activities.     

Antibacterial peptides from barbel muscle protein hydrolysates: Activity against some pathogenic bacteria

Peptides obtained by enzymatic hydrolysis of fish proteins exhibit not only nutritional but also biological properties of dietary uses, or even therapeutic potential. The objective of the present study was to isolate and characterize peptides from the protein hydrolysates of barbel muscle with antibacterial activity against Gram-positive (Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus and Bacillus cereus) and Gram-negative (Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter sp.) bacteria. Barbel muscle protein hydrolysates (BMPHs), obtained by treatment with Alcalase^® (DH = 6.6%), was fractionated by size exclusion chromatography on a Sephadex G-25 and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses and amino acid sequences of these peptides were determined using ESI–MS and ESI–MS/MS, respectively. Eleven peptides in F_II-1, F_II-2, F_II-3 and F_II-4 sub-fractions separated by RP-HPLC were identified. The most active peptide fraction (F_II-3) contained three peptides: Ala–Ala–Ala–Leu; Ala–Ala–Gly–Gly–Val and Ala–Ala–Val–Lys–Met.     

SUBSTRATE SPECIFICITY OF AMINOPEPTIDASE FROM JAPANESE CLASSIFIED BARLEY FLOUR

In this study, some properties of a peptidase obtained from Japanese barley were investigated. Boc‐Val‐Leu‐Lys‐MCA was slightly hydrolyzed, but the enzyme showed almost no activity on Suc‐Ala‐Ala‐Pro‐Phe‐MCA and Ac‐Val‐Glu‐Ile‐Asp‐MCA. The enzyme activity decreased to 57.7% by the addition of 0.235 mM bestatin. All tripeptides and tetrapeptides studied were cleaved from the N‐terminus amino acid by the enzyme. However, the enzyme did not cleave Leu‐Pro‐Phe‐Phe‐Asp in the manner of the AP type. The C‐terminus amino acid residue (Asp) and the second amino acid residue of the C‐terminus (Phe) were released at almost the same time. From these findings, this enzyme was identified as both an AP and oligopeptidase. This property is very similar to that of cathepsin H and the novel peptidase purified from mesquite pollen. This enzyme may not only supply useful foodstuffs such as a meat tenderizer but also produce bioactive peptides.     

Diketopiperazines in wines

This study describes the identification of cyclic dipeptides known as diketopiperazines in red, white and sparkling wines from Greek market. Diketopiperazines have been isolated from bottled wines after liquid–liquid extraction and identified using gas chromatography coupled with mass spectrometry. Cyclo (Leu–Leu), cyclo (Leu–Pro), cyclo (Phe–Pro), cyclo (Leu–Phe), cyclo (Val–Phe) and cyclo (Ala–Phe) were the six diketopiperazines that were identified in bottled wines made from local and international plant varieties. The varieties tested were agiorgitiko, malagouzia, xinomauro, roditis, muscat, athiri, mandilari, vidiano, cabernet sauvignon, syrah and merlot. The cyclo (Leu–Pro) was identified in eighteen bottled wines while the cyclo (Phe–Pro), cyclo (Leu–Phe), cyclo (Leu–Leu), cyclo (Ala–Phe) and cyclo (Val–Phe) were identified in a small number of samples. The analysis of some wine samples showed that the values of cyclo (Leu–Pro) ranged from 0.1 to 1?mg/L. These cyclic dipeptides have biological properties and may contribute to the beneficial health effects of wine.     

Simultaneous addition of Palatase M and Protease P to a dry fermented sausage (Chorizo de Pamplona) elaboration: Effect over peptidic and Lipid fractions

The effect of the simultaneous addition of a lipase (Palatase M 200L Novo Nordisk A/S) and a protease (Protease P 31.000 Solvay Enzymes GMBH&CO.KG) to the manufacture of a Spanish dry fermented sausage (Chorizo de Pamplona) was studied. In relation to the free aminoacid fraction, significative increases in Glu, His, Lys, Ser, Ala, Pro, Val, Met, Ile, Leu and Phe were found. Smaller differences were observed in the aminoacids obtained from peptides. The addition of the lipase caused significant increases in palmitic, oleic and linolenic acids. Despite the observed changes, no differences were found in the sensory quality compared with the control, except for a slight softening.     

5种氨基酸热失重行为及其热解生成氢氰酸的研究

采用改造后的商品化热重分析仪,对不同升温速率和裂解气氛下甘氨酸(Gly)、丙氨酸(Ala)、亮氨酸(Leu)、异亮氨酸(Ile)、脯氨酸(Pro)等5种氨基酸热解特性及生成氢氰酸(HCN)的行为进行了研究。结果表明:①除Gly外,其他4种氨基酸生成HCN的产率随着升温速率的提高而增加;②当有O2存在时,HCN的产率较N2裂解气氛下有明显降低,当O2含量达到20%时,5种氨基酸生成HCN的产率均低于0.25%;③在不同的升温速率和裂解气氛下,Gly生成HCN的产率均高于其他4种氨基酸;在10%O2+90%N2气氛和40℃/min的升温速率下,5种氨基酸生成HCN产率顺序:Gly>Leu>Ile>Pro>Ala。     

Multifunctional peptides from egg white lysozyme

Hen’s egg white lysozyme (HEWL) is one of the major egg white proteins with well demonstrated antimicrobial activity. Bioactive peptides other than antimicrobial peptides from HEWL have not been reported; therefore, the purpose of the study was to explore new bioactivities of lysozyme-derived bioactive peptides. HEWL was hydrolysed with Alcalase and fractionated by cation-exchange chromatography. The Alcalase HEWL hydrolysate and its fractions were analyzed for inhibitory activities against calmodulin-dependent phosphodiesterase (CaMPDE) and antioxidant activities using oxygen radical absorbance capacity-fluorescein (ORAC-FL), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS⁺) and 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) radical scavenging methods. The fractionated peptides had higher CaMPDE inhibition activity, ORAC-FL value and ABTS⁺ scavenging activity than those of the hydrolysate. Peptide sequences in the most overall active fractions were characterized by LC–MS/MS. Our results showed that HEWL hydrolysate and its peptide fractions may serve as useful ingredients in the formulation of functional foods and nutraceuticals.     

Comparative study of partial amino acid sequences of prolamins and glutelins from various cereals. VII. Amino acid sequences of prolamin peptides

The peptide fractions isolated from chymotryptic hydrolysates of wheat, rye and barley prolamines [this journal (1984) 178:173] were separated into pure peptides by high-performance liquid chromatography on octadecyl silica gel. The peptides which were most abundant were analyzed for amino acid composition and in part for amino acid sequence. Besides peptides which are typical for only one of the cereals investigated, peptides of similar composition were found in all three prolamines. These contain repeating sequences and are built up of mainly Gln (Q), Pro (P) and hydrophobic amino acids (X) such as Phe, Tyr, Ile, Val and Leu. One of the most frequent partial sequences is QQPQQPXP.