Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis

The hydrolysis of crude egg white with pepsin, trypsin, and chymotrypsin produced peptides with angiotensin-converting enzyme (ACE) inhibitory properties. These peptides were mainly derived from the proteolysis of ovalbumin. The most active hydrolysates were obtained after treatment with pepsin (50% inhibitory concentration [IC50], 55.3 microg/ml), with the fraction having a molecular mass lower than 3,000 Da giving the highest ACE inhibitory activity (IC50, 34.5 microg/ml). Nine subfractions were collected from the fraction with a molecular mass lower than 3,000 Da using semipreparative reversed-phase high-performance liquid chromatography. Considerable ACE inhibitory activity (IC50 < 40 microg/ml) was found in three of them. These subfractions were analyzed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry, and 14 peptides were identified. These sequences were synthesized, and their ACE inhibitory activities were measured. Among the identified peptides, two novel sequences with potent ACE inhibitory activity were found. The amino acid sequences of these inhibitors were identified as Arg-Ala-Asp-His-Pro-Phe-Leu and Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu and showed IC50 values of 6.2 and 4.7 microM, respectively.     

酪朊酸钠制备ACE抑制肽的研究

采用商业化的胰蛋白酶对底物浓度为10％的酪朊酸钠进行可控酶解，将酶解液的上清液经截留分子量为10ku的超滤膜分离后利川紫外分光光度法测定各组分ACE体外抑制活性，结果表明酪朊酸钠的胰蛋白酶酶解液具有较强的ACE抑制活性，超滤分离可提高产品的ACE体外抑制活性，为开发新一代降血压保健食品提供广阔的前景。     

Inhibition of the in vitro activities of α-amylase, α-glucosidase and pancreatic lipase by yellow field pea (Pisum sativum L.) protein hydrolysates

The aim of this work was to produce yellow field pea protein-derived peptides as inhibitors of α-amylase, α-glucosidase and pancreatic lipase activities. A pea protein concentrate was hydrolysed with alcalase, chymotrypsin, pepsin or trypsin and the hydrolysates separated into different fractions (<1, 1–3, 3–5, 5–10 kDa) by membrane ultrafiltration. Peptide sequence analysis showed that the alcalase hydrolysate had higher levels of di- and tripeptides when compared with the chymotrypsin, pepsin and trypsin hydrolysates. The peptide fractions inhibited α-amylase and α-glucosidase activities at levels that were similar to the unfractionated hydrolysates. The peptides were more active against α-amylase (inhibition at μg level) than α-glucosidase (mg level). In contrast, the fractionated peptides had reduced ability (IC₅₀ >4.2 mg mL⁻¹) when compared with the unfractionated hydrolysate (IC₅₀ <4.2 mg mL⁻¹) to inhibit lipase activity. Enzyme kinetic studies revealed that the peptides reduced α-amylase activity through competitive inhibition. However, inhibition of α-glucosidase activity was non-competitive.     

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.     

Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: Effect of substrate,enzyme and time of hydrolysis

Nine milk protein substrates were hydrolysed in vitro with five proteases for various times (0, 3, 6, and 24 h), and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates was assessed. Overall, the casein substrates gave rise to hydrolysates with significantly higher ACE-inhibitory activity than the whey protein (WP) substrates (85% vs. 79%). No significant difference between 3 and 24 h of hydrolysis was found. A reasonable correlation was found between the ACE inhibition of the 6 h hydrolysates determined in vitro and estimated by in silico modelling. The highest ACE-inhibitory activity was found in hydrolysates made with thermolysin followed by proteinase K, trypsin, pepsin and Bacillus licheniformis protease. The IC₅₀ values for thermolysin hydrolysates of caseins and WPs were 45–83 and 90–400 μg mL⁻¹, respectively, with α-lactalbumin giving the highest inhibitory activity. Thermolysin, proteinase K and trypsin were useful for the release of highly potent ACE-inhibitory peptides from both WPs and caseins.     

Novel angiotensin-I-converting enzyme inhibitory peptides derived from recombinant human alpha s1-casein expressed in Escherichia coli.

Recombinant human alpha s1-casein expressed in Escherichia coli was purified and digested with trypsin in an attempt to find peptides with angiotensin-I-converting enzyme (ACE) inhibitory activity. Three novel ACE inhibitory peptides, A-II, B-II and C, were isolated and their amino acid sequences identified as Tyr-Pro-Glu-Arg (residues 8-11), Tyr-Tyr-Pro-Gln-Ile-Met-Gln-Tyr (residues 136-143) and Asn-Asn-Val-Met-Leu-Gln-Trp (residues 164-170) respectively. ACE inhibitory activities were measured for the corresponding synthetic peptides, and the ACE IC50 (the amount of peptide causing 50% inhibition of ACE activity) values of A-II, B-II and C estimated to be 132.5, 24.8 and 41.0 mumol/l respectively. Peptides A-II and C were resistant to further digestion by pepsin, whereas peptide B-II was hydrolysed. All three peptides were resistant to digestion by chymotrypsin. These ACE inhibitory peptides may prove useful for oral administration in the treatment of hypertension.     

Angiotensin I converting enzyme-inhibitory activity of bovine, ovine, and caprine kappa-casein macropeptides and their tryptic hydrolysates

This work evaluated the angiotensin-converting enzyme (ACE)-inhibitory activities of bovine, ovine, and caprine kappa-casein macropeptides (CMPs) and their tryptic hydrolysates. The results obtained indicate that bovine, ovine, and caprine CMPs exhibited moderate in vitro ACE-inhibitory activities that increased considerably after digestion under simulated gastrointestinal conditions. Active peptides could also be produced from CMPs via proteolysis with trypsin, with tryptic hydrolysates exhibiting a more extensive ACE-inhibitory activity than intact CMPs during simulated gastrointestinal digestion. Two active fractions were chromatographically separated from the tryptic hydrolysate of the bovine CMP, but their complexity hampered the assignment of the ACE-inhibitory activity to specific peptide sequences. Evidence for the release of the strong ACE-inhibitory tripeptide IPP was found upon simulation of the gastrointestinal digestion of peptides released by trypsin from the CMP sequence. These findings might help to promote further exploitation of cheese whey in the preparation of nutraceuticals for inclusion in the composition of functional food products with high added values.     

Evaluation of angiotensin I-converting enzyme (ACE) inhibitory activities of hydrolysates generated from byproducts of freshwater clam

Solid wastes of freshwater clam in food processing, including mainly mantle, were used as a raw material for the recovery of bioactive peptides related to angiotensin I-converting enzyme (ACE) inhibitor. Among the primary hydrolysates of dried mantle (DM), the peptides corresponding to hydrolysates using 2 crude peptidases exhibited a strong ACE inhibitory activity (IC₅₀, 0.23 mg/mL), and recovery efficiency of soluble materials and their protein content were considerably large with 42.65% and 468.6 mg/g, respectively. The ACE inhibitory activity of all secondary hydrolysates digested by pepsin and trypsin was significantly increased as compared to primary hydrolysates. Furthermore, the peptic secondary hydrolysates were fractionated by gel filtration and reverse phase-HPLC (RP-HPLC) and characterized by matrix-assisted laser desorption-ionization time of flight/mass spectrometry (MALDI-TOF/MS). These peptides with molecular weight of less than 1 kDa possessed the stronger ACE inhibitory effect, and their inhibitory pattern was found to be competitive. The results showed that the DM hydoplysates might be utilized as a rich source of bioactive peptide.     

螺旋藻蛋白水解产物对ACE抑制活性的研究

采用胃蛋白酶和胰蛋白酶水解螺旋藻藻胆蛋白制备ACE 抑制肽,通过体外实验测定其ACE 抑制率,以ACE 抑制率为指标确定两种蛋白酶的水解条件。结果表明：胃蛋白酶水解条件为：水解温度37℃,酶与底物比1:50,pH3.0,底物质量分数5%,水解产物的ACE 抑制率为82.16%,其IC50 值为0.104mg/ml;胰蛋白酶水解条件为：水解温度42℃,酶与底物1:50,pH8.0,底物质量分数6%,水解产物的ACE 抑制率为93.54%,其IC50值为0.017mg/ml。此外,胃蛋白酶水解产物再用胰蛋白酶水解,其产物的IC50 值为0.087mg/ml。     

Preparation of angiotensin-I-converting enzyme inhibitory hydrolysates from unsupplemented caprine whey fermentation by various cheese microflora

Unsupplemented caprine whey was fermented by 25 cheese microflora in order to produce peptides from α-lactalbumin (α-la) and/or β-lactoglobulin (β-lg) hydrolysis. Fourteen hydrolysates enriched in peptides mainly released from α-la were obtained. Angiotensin-I-converting enzyme (ACE) inhibitory activity of each hydrolysate was investigated. Six of them had high ACE inhibitory activities ranging from 31% to 56%. The highest ACE inhibitory activity was obtained after whey fermentation by the microflora from 18 months ripened Comté cheese. The microflora was identified as a co-culture of Candida parapsilosis and Lactobacillus paracasei. Hydrolysate activity remained stable after pepsin, trypsin and chymotrypsin treatments simulating an in vitro gastrointestinal digestion. This hydrolysate was further fractionated by RP-HPLC. The peptide exhibiting the highest ACE inhibitory activity was characterised as WLAHK (α-la f(104–108): Trp–Leu–Ala–His–Lys). WLAHK was resistant toward pepsin and trypsin treatments but was digested by chymotrypsin.     

Angiotensin-I converting enzyme inhibitory and antioxidant activities of egg protein hydrolysates produced with gastrointestinal and nongastrointestinal enzymes

Egg is a well-known rich source of bioactive peptides. In this study, egg protein (egg white and egg yolk proteins) hydrolysates were produced with gastrointestinal enzymes (pepsin and pancreatin) or nongastrointestinal enzymes (thermolysin and alcalase), and fractionated by ultrafiltration and cation exchange chromatography. Angiotensin-I converting enzyme (ACE) inhibitory and antioxidant activities, amino acid composition and molecular weight distribution were studied, and the physicochemical properties were related with the bioactivities. Our results showed that egg protein hydrolysates produced with non-GI enzymes (thermolysin and alcalase) showed significantly higher ACE inhibitory activity, whereas similar or even lower antioxidative activities, than those of hydrolysates produced with GI enzymes. ACE-inhibitory activity significantly correlated with the amino acid composition, especially the proportion of positively charged amino acid, whereas antioxidant activities correlated with the proportion of low molecular weight peptides under 500 Da. Understanding the relationship between the bioactivities and physicochemical properties of the hydrolysates/fractions is important to facilitate the development technologies for preparing fractions with improved bioactivities.     

菠萝蛋白酶水解泥鳅蛋白制备ACE抑制肽的研究

为了探讨利用泥鳅蛋白制备功能性肽的可能性,采用高效液相色谱法测定泥鳅肉水解物对血管紧张素转换酶(ACE)的抑制作用,从胰蛋白酶、胃蛋白酶、菠萝蛋白酶、复合风味蛋白酶、复合蛋白酶5种酶中筛选出菠萝蛋白酶作为酶解泥鳅肉制备具有降血压活性水解物的适宜水解酶。在单因素试验的基础上,采用L9(34)正交试验设计对该酶的酶解条件进行优化。结果表明最佳水解条件为：温度55℃,固液比1:3,pH6.5,加酶量1000U/g pro,水解时间90min。在该条件下,水解物的ACE抑制率IC50值为0.0184mg/mL,ACE抑制肽的相对分子质量主要集中在924左右。     

杏仁蛋白水解物对血管紧张素转化酶抑制作用的研究

分别采用Protamex、Alcalase、Neutrase、Flavourzyme、Proleather FG-F、木瓜蛋白酶水解杏仁蛋白,利用高效液相色谱法测定水解物对血管紧张素转化酶(ACE)的抑制活性,以水解度(DH)和水解产物对ACE的抑制率为指标对酶解过程进行分析,并研究水解物的体外消化稳定性。结果表明,Proleather FG-F 和Alcalase 对杏仁蛋白有较好的水解效果,其水解物对ACE 抑制率较高,IC50 分别为1.24mg/ml 和0.98mg/ml。模拟胃肠消化实验结果表明,在消化酶的作用下杏仁蛋白水解物仍具有较强的ACE 抑制活性。     

Effect of simulated gastrointestinal digestion on the antihypertensive properties of synthetic beta-lactoglobulin peptide sequences

In this study, the antihypertensive activity in spontaneously hypertensive rats of two peptides isolated from beta-lactoglobulin hydrolysates with thermolysin was evaluated. These peptides, with sequences LLF [beta-lg f(103-105)] and LQKW [beta-lg f(58-61)], showed potent in vitro ACE-inhibitory activity. Two hours after administration, both sequences caused a clear and significant decrease in the blood pressure of these rats. The impact of a simulated gastrointestinal digestion on ACE-inhibitory and antihypertensive activities of these peptides was also studied. The results showed that both fragments were susceptible to proteolytic degradation after incubation with pepsin and Corolase PP. In addition, their in vitro ACE-inhibitory activity decreased after the simulated digestion. It is likely that fragment LQK was the active end product of the gastrointestinal digestion of peptide LQKW. The fragment LL, observed after digestion of peptide LLF, probably exert its antihypertensive effect through a mechanism of action different than ACE-inhibition.     

Purification of goat beta-lactoglobulin from whey by an ultrafiltration membrane enzymic reactor

This paper presents a novel contribution to the purification of goat beta-lactoglobulin by using an ultrafiltration membrane enzymic reactor. The basis of the purification process was the enzymic hydrolysis of contaminating proteins, alpha-lactalbumin and traces of serum albumin, by pepsin at 40 degrees C and pH 2, conditions under which beta-lactoglobulin is resistant to peptic digestion. Simultaneously, beta-lactoglobulin and peptides were separated by ultrafiltration. beta-Lactoglobulin was retained in the reactor while peptides generated by hydrolysis from alpha-lactalbumin and serum albumin permeated through the membrane. The process was made continuous by the addition of fresh whey to replace the lost permeate. Three mineral membranes with 10, 30 and 50 kDa molecular mass cut-off were tested and the 30 kDa membrane was selected for the continuous process. The simultaneous purification and concentration of beta-lactoglobulin from clarified goats' whey was achieved in a single step. The ultrafiltration membrane enzymic reactor could treat eight reactor volumes of clarified whey. The recovery of beta-lactoglobulin was 74%, its purity was 84% and its concentration 6.6-fold that in the initial clarified whey.     

Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase

Peptides from hydrolysates of fish proteins and from cheeses were analysed for inhibition of prolyl endopeptidase (PE) isolated from porcine muscle. Muscles of cod, salmon, and trout were homogenised and incubated at pH 4.0 with pepsin and then at pH 7.5 with trypsin to obtain fish protein hydrolysates. Homogenates were incubated without exogenous enzymes at pH 4.0 and 7.5 to obtain fish protein autolysates. Water-soluble extracts from "rakfisk" (a Norwegian fermented/autolysed trout muscle dish) and water-soluble extracts from Cheddar, Norvegia, Jarlsberg, and Blue cheese were also prepared. Peptides in the supernatants obtained after heat-treatment of fish hydrolysates, autolysates and water-soluble extracts of rakfisk and cheeses at 95 degrees C for 15 min were analysed for inhibition of PE. Inhibition was also measured in peptide fractions separated by reversed-phase high-performance chromatography and by gel permeation chromatography. The peptide fractions from fish hydrolysates, fish autolysates, and water-soluble extracts of cheeses inhibited PE in hydrolysing Z-Gly-Pro-amidomethylcoumarin. Inhibition by peptides from rakfisk was negligible. Pepsin + trypsin hydrolysates from the three fish species contained PE inhibitory peptides with a broad range of apparent hydrophobicity and apparent molecular mass. Autolysates from muscles of the 3 fish species contained narrow peptide peaks of different molecular mass and different apparent hydrophobicity with strong PE inhibitory activity. The content of hydrophilic inhibitory peptides was lower in cheeses than in pepsin + trypsin hydrolysates of fish muscle.     

酪蛋白双酶水解物ACE抑制肽的分离纯化

采用胃蛋白酶和胰蛋白酶依次对酪蛋白进行双酶水解,制备ACE抑制肽。水解物经截留分子质量6ku的超滤膜初步分离,再通过Sephadex G-15进一步纯化,体外测定各分离产物ACE活性的半数抑制质量浓度(IC50值)。纯化得到的各组分经毛细管电泳分析肽谱、Q-TOF LC/MS检测分子质量范围。结果显示：双酶水解产物IC50值为560μg/mL,超滤流出物IC50值为250μg/mL;Sephadex G-15分离得到3个组分,组分Ⅰ的IC50值为123.41μg/mL,含有19个肽段;组分Ⅱ的IC50值为66.67μg/mL,含有14个肽段;组分Ⅲ的IC50值为64.29μg/mL,含有5个肽段。Q-TOF LC/MS测得纯化组分的分子质量范围为400～800u。     

Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate

Our objective was to evaluate the angiotensin I converting enzyme (ACE) inhibitory activity of skate skin protein hydrolysates and its corresponding fractions. The skate skin hydrolysates were obtained by enzymatic hydrolysis using alcalase, α-chymotrypsin, neutrase, pepsin, papain, and trypsin. Amongst the six hydrolysates, the α-chymotrypsin hydrolysate had the highest ACE inhibitory activity compared to other hydrolysates. The amino acid sequences of the purified peptides were identified to be Pro–Gly–Pro–Leu–Gly–Leu–Thr–Gly–Pro (975.38 Da), and Gln–Leu–Gly–Phe–Leu–Gly–Pro–Arg (874.45 Da). The purified peptides from skate skin had an IC₅₀ value of 95 μM and 148 μM, respectively, and the Lineweaver–Burk plots suggest that they act as a non-competitive inhibitor against ACE. Our study suggested that novel ACE inhibitory peptides derived from skate skin protein may be beneficial as anti-hypertension compounds in functional foods.     

Preparation and antioxidant activity of enzymatic hydrolysates from purple sea urchin (Strongylocentrotus nudus) gonad

Purple sea urchin (Strongylocentrotus nudus) gonad was treated separately with neutral protease, papain, pepsin and trypsin. The resultant hydrolysates were fractionated using a series of ultrafiltration membranes (molecular weight cut-offs of 10, 5, 3 and 1 kDa). Five fractions were prepared from each hydrolysate and the corresponding molecular weight ranges were below 10 kDa, 5-10 kDa, 3-5 kDa, 1-3 kDa and below 1 kDa. The peptide fractions were evaluated for antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and reducing power assay. Results indicated that all peptide fractions possessed DPPH radical scavenging capacity and reducing power in a dose-dependent manner. For all four hydrolysates, the below 1 kDa fractions exhibited the highest DPPH radical scavenging capacity. The below 1 kDa fractions prepared with neutral protease, papain and pepsin, and the 1-3 kDa fraction prepared with trypsin showed the highest reducing capacity among corresponding hydrolysates.     

Indigenous marine diatoms as novel sources of bioactive peptides with antihypertensive and antioxidant properties

Several bioactive compounds from microalgae have demonstrated diverse biological activities with positive effects on human health. However, the potential of bioactive peptides as functional foods is still undervalued. Therefore, the exploration of microalgae strains as sources of bioactive peptides could reveal strong and unique bioactivities, especially when these marine sources have never been explored before. For this aim, protein extracts from six indigenous marine diatoms were subjected to enzymatic hydrolysis using four proteases (flavourzyme, pepsin, papain and trypsin). The hydrolysates were then tested for angiotensin converting enzyme (ACE)-inhibitory, antioxidant and antihypertensive properties. Results showed that papain hydrolysates from all microalgae strains exhibited strong ACE-inhibitory activities and antioxidant properties. In particular, protein hydrolysates from Bellerochea malleus were found to reduce blood pressure properties of 17 mmHg after 5 days of oral administration to SHR animals. These results revealed the potential of bioactive peptides from indigenous marine diatoms for use as functional foods or nutraceuticals.