Antihypertensive effect of bioactive peptides produced by protease-facilitated lactic acid fermentation of milk

Milk was fermented for up to 5 h at 43 °C with two lactic acid bacteria (Streptococcus thermophilus, Lactobacillus bulgaricus). A protease, flavourzyme, was added at the beginning of fermentation. The whey fraction was separated from the fermented milk and freeze-dried. During the 5 h of fermentation, the soluble protein content increased from 4.9 to 57.4 mg/g and peptide content increased from 2.1 to 32.8 mg/g, while inhibition of angiotensin I-converting enzyme (ACE) increased by a decrease of IC₅₀ from 0.708 to 0.266 mg/ml, respectively. The whey was fractionated into four fractions by size exclusion chromatography on a Sephadex G-15 column. The fourth fraction of the whey showed the highest inhibitory efficiency ratio (IER) being 1329%/mg/ml. The amino acid sequence of the inhibitory peptide was Tyr-Pro-Tyr-Tyr, of which the IC₅₀ was 90.9 μM. The whey showed mixed-type inhibition kinetics, while Captopril, the positive control showed competitive inhibition on ACE. Their K_i values were 0.188 mg/ml and 0.0067 μg/ml, respectively. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) was reduced to 15.9 and 15.6 mm Hg, respectively, in spontaneously hypertensive rat (SHR), after 8 weeks of oral administration of diluted whey (peptide concentration 4.9 mg/ml).     

Antihypertensive effect of a bovine lactoferrin pepsin hydrolysate: Identification of novel active peptides

The potential of bovine lactoferrin (LF) as a source of antihypertensive peptides has been examined. For this purpose, LF pepsin hydrolysate with molecular mass lower than 3 kDa (LFH < 3 kDa) was prepared and orally administered to spontaneously hypertensive rats (SHR), resulting in reduced systolic blood pressure in a significant and maintained manner up to 24 h after administration. LFH < 3 kDa was further fractionated by semi-preparative high performance liquid chromatography (HPLC) and 38 peptides, contained in the active fractions, were identified by using an ion trap mass spectrometer. Based on the peptide abundance, a total of 11 peptides were chemically synthesized and their ACE inhibitory activity tested. Only three of them, corresponding to peptides of sequences LIWKL, RPYL and LNNSRAP exerted in vitro inhibitory effects on angiotensin I converting enzyme (ACE) activity and had a 50% inhibitory concentration (IC₅₀) of 0.47, 56.5 and 105.3 μM, respectively. The three peptides also showed antihypertensive effects in SHR and remarkably the effect of LIWKL remained significant for up to 24 h post-administration, similarly LFH < 3 kDa and the captopril control. The two most potent in vitro inhibitory peptides showed ex vivo inhibitory effect on ACE-dependent vasoconstriction as well. In conclusion, three novel LF-derived peptides and a pepsin LFH < 3 kDa lowered blood pressure and exhibit potential as orally effective antihypertensive compounds.     

A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats

Tuna frame protein was hydrolysed using Alcalase, Neutrase, pepsin, papain, α-chymotrypsin and trypsin. Peptic hydrolysate exhibited the highest ACE I inhibitory activity among them and was fractionated into three ranges of molecular weight (below 1, 1–5 and 5–10 kDa) using an ultrafiltration membrane bioreactor system. The 1–5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from tuna frame protein (PTFP), which was composed of 21 amino acids, Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-Val-Ser-Asn-Trp-Ser-Pro-Pro-Lys-Try-Lys-Asp-Thr-Pro (MW: 2,482 Da, IC₅₀: 11.28 μm), was isolated. Lineweaver–Burk plots suggest that PTFP plays as a non-competitive inhibitor against ACE. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHR) also revealed that oral administration of PTFP can decrease systolic blood pressure significantly (P < 0.01). These results suggest that the PTFP would be a beneficial ingredient for nutraceuticals and pharmaceuticals against hypertension and its related diseases.     

Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC–ESI-TOF MS/MS

Haruan myofibrillar protein was hydrolysed with proteinase K and thermolysin to isolate Angiotensin converting enzyme (ACE) inhibitory peptides. The thermolysin hydrolysate of myofibrillar protein with the highest ACE inhibition activity (IC₅₀ = 0.033 mg/ml) was fractionated by ultrafiltration and size exclusion chromatography to three fractions. Fraction F2 with higher ACE inhibitory activity was separated into five fractions (A–E) using reversed-phased high performance liquid chromatography (RP-HPLC). Fraction C showed 81% inhibition activity and was subjected to HPLC coupled to electrospray ionisation-time-of-flight mass spectrometry (ESI-TOF MS/MS). Two peptide sequences for the most abundant fragments were identified as VPAAPPK (IC₅₀ = 0.45 μM) at 791.155 m/z and NGTWFEPP (IC₅₀ = 0.63 μM) at 1085.841 m/z. The presence of two proline residues at the C-terminal sequence is responsible for the high ACE inhibitory activity of these peptides. The results suggest that Haruan meat protein hydrolysate is a potent ACE inhibitor and may be used to decrease blood pressure.     

Identification and characterization of novel angiotensin-converting enzyme inhibitors obtained from goat milk

The aim of the present study was to identify and characterize new inhibitory peptides of angiotensin I-converting enzyme (ACE) from goat milk and to analyze the effect of long-term intake of a goat milk hydrolysate-supplemented (GP-hyd) diet on the development of hypertension in spontaneously hypertensive rats (SHR). Three new inhibitory peptides for ACE (TGPIPN, SLPQ, and SQPK) were isolated. The inhibitory concentration 50% (IC₅₀) values of individual peptides were 316, 330, and 354 μmol/L, respectively. Only TGPIPN was found to pass intact a monolayer of Caco-2 cells in small amounts. The SHR fed for 12 wk a diet (GP-hyd) enriched in a hydrolysate containing these peptides (estimated intake of TGPIPN was 230 mg/kg per d) showed lower (approximately 15 mmHg) systolic blood pressure than animals fed a control diet. The ACE activities in the aorta, left ventricle, and kidney were significantly decreased in the GP-hyd group compared with those of the control group and were similar to those found in SHR fed captopril (130 mg/kg per d). Impaired endothelium-dependent relaxation to acetylcholine by aortic rings from SHR was improved in those fed the GP-hyd diet. The left ventricle weight and kidney weight index were significantly reduced in the GP-hyd group and captopril groups. Moreover, long-term treatment of SHR with a diet enriched in goat milk hydrolysate, or captopril, attenuated the development of hypertension, cardiac and renal hypertrophy, and endothelial dysfunction. These effects might be related to the in vivo inhibitory effects of the hydrolysate on tissue ACE activity.     

Production,in continuous enzymatic membrane reactor,of an anti-hypertensive hydrolysate from an industrial alfalfa white protein concentrate exhibiting ACE inhibitory and opioid activities

A few hydrolysates of food proteins were reported to exert an antihypertensive effect in vivo due to angiotensin I converting enzyme (ACE) inhibitory peptides or opioid peptides. An industrial alfalfa white protein concentrate (AWPC) hydrolysate, produced on a pilot plant scale, strongly inhibited ACE with an IC50 value of 8.8 μg/ml. Furthermore, the AWPC hydrolysate inhibited the spontaneous contraction of rabbit ileum in a dose-dependent and naloxone-blocked manner, indicating the presence of opioid peptides. The AWPC hydrolysate significantly lowered blood pressure of spontaneously hypertensive rats (SHR) more than 6 h after a single oral administration. The maximum reduction of systolic blood pressure (−29.9 ± 2.0 mmHg) was observed 4 h after an oral administration of 0.5 g/kg hydrolysate dosage. The minimal anti-hypertensive effect was observed for 0.1 g/kg dosage. The AWPC hydrolysate may then be particularly suitable as nutraceutical for functional food useful in the prevention of hypertension.     

Amaranth seed protein hydrolysates have in vivo and in vitro antihypertensive activity

The objective of this work was to study the hydrolytic release of encrypted peptides with antihypertensive activity from storage proteins of Amaranthus mantegazzianus, as determined by in vitro assays, for the first time by in vivo studies in animal models, and by ex vivo assays. Hydrolysates with hydrolysis degree (DH) of 45% and 65% (IC50 0.12 mg/ml, equivalent to 300–600 μM) exhibited an angiotensin-I converting enzyme 1 (ACE) inhibitory activity equal or higher than the potential inhibitory of the average antihypertensive peptides registered in the BIOPEP database and of semi-purified Amaranthus hypochondriacus albumin and globulin protein fractions. Intragastric administration of hydrolysates with DH of 45% was effective in lowering blood pressure of male spontaneously hypertensive rats (SHR). Experiments performed in papillary muscles isolated from hearts and with isolated aortic smooth muscle of SHR suggest that the hypotensive effect could be attributed to a lowering of the peripheral resistance. We assume that the amaranth hydrolysates would be acting at the level of the local or autocrine renin–angiotensin system (RAS).     

Novel angiotensin I-converting enzyme inhibitory peptide derived from bovine casein

Bovine lactic casein was hydrolysed using a combination of three enzymes, namely, subtilisin, bacillolysin, and trypsin, and the resulting preparation was coined CH-3. CH-3 showed angiotensin I-converting enzyme (ACE)-inhibitory activity (IC₅₀: 74 μg/mL). A single oral administration of CH-3 led to a transient but significant decrease in the systolic blood pressure (SBP) of spontaneously hypertensive rats (SHRs), while daily oral administration of CH-3 for 28 consecutive days led to a lower rate of SBP increase. The CH-3 preparation was then fractionated and the _αS2-casein-derived tripeptide Met-Lys-Pro (or MKP) was identified as a novel peptide with strong ACE-inhibitory activity (IC₅₀ = 0.12 μg/mL, 0.3 μM). The MKP peptide constituted only 0.053% of CH-3 but its activity was accounted for 33% of the total ACE-inhibitory activity of CH-3. In addition, a single oral administration of MKP also led to a transient but significant decrease in the SBP of SHRs.     

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 (6 kDa < HSSG-I < 10 kDa, 2 kDa < HSSG-II < 6 kDa, HSSG-III < 2 kDa) using an ultrafiltration unit. The HSSG-III showed the most potent ACE inhibitory activity in vitro with IC₅₀ of 0.33 mg/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 30 days 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.     

Novel angiotensin I-converting enzyme inhibitory peptides derived from soya milk

Inhibitors of angiotensin I-converting enzyme (ACE) are useful in treating hypertension, and many have been derived from food products, including soybeans. Using the industrial protease PROTIN SD-NY10, we developed a processed soya milk (PSM) with enhanced ACE inhibitory activity. The ACE inhibitory activity of PSM (IC₅₀ = 0.26 μg/ml) was greater than that of regular soya milk (IC₅₀ = 8.75 μg/ml). Eight novel ACE inhibitory peptides were purified from PSM by reversed-phase chromatography: FFYY (IC₅₀,1.9 μM), WHP (4.8 μM), FVP (10.1 μM), LHPGDAQR (10.3 μM), IAV (27.0 μM), VNP (32.5 μM), LEPP (100.1 μM), and WNPR (880.0 μM). The IC₅₀ values of these peptides are comparable to those reported for other ACE inhibitory peptides derived from wheat, chicken, bonito, wine, etc. Due to the relatively low IC₅₀ values of several peptides identified in this study, they may serve as ideal base components of food supplements for patients with hypertension.     

米糠蛋白ACE抑制肽在大鼠体内降压效果的研究

采用灌胃和静脉注射的方法,观察了米糠蛋白ACE抑制肽对应激性高血压(SHR)大鼠的降压效果,并与卡托普利的降压效果进行了比较。分别以50、100、150 mg/kg·bw剂量的米糠蛋白ACE抑制肽一次性给药后,每2 h测量大鼠血压,连续测量8 h;实验持续4周,每天灌胃一次,每周测量血压及体重一次。SHR大鼠给予米糠蛋白ACE抑制肽后,血压均显著下降(p<0.05),并均在灌胃后2 h达到最低值,血压下降最大幅度为(31±6.8)mm Hg;而正常大鼠灌胃米糠蛋白ACE抑制肽后血压无显著变化,灌胃卡托普利则会明显降低其血压;长期灌胃米糠蛋白ACE抑制肽可使SHR大鼠血压下降明显(p<0.05),且降压效果稳定。      

鮰鱼皮明胶ACE抑制肽降血压活性的研究

鮰鱼皮为斑点叉尾鮰鱼片加工的主要副产物,利用鮰鱼皮明胶制备降血压肽(ACE抑制肽)可为鮰鱼皮的高值化利用提供参考。本文通过对原发性高血压大鼠(SHR)进行一次性及长期给药(28 d)实验,研究鮰鱼皮明胶ACE抑制肽(Mr3000 Da)的降血压作用,并测定大鼠血清和肺组织中的ACE活性和Ang II含量。实验结果表明:一次性给药和长期灌胃给药鮰鱼皮明胶ACE抑制肽均对SHR大鼠有显著降血压作用,降血压效果呈剂量依赖性;高剂量组SHR大鼠灌胃给药2 h后,其收缩压由206 mmHg降至159 mmHg;高剂量组SHR大鼠经长期灌胃给药10 d后,血压一直保持在155 mmHg左右;同时,鮰鱼皮明胶ACE抑制肽对正常血压大鼠的降血压作用不显著。鮰鱼皮明胶ACE抑制肽对SHR大鼠血清和肺组织中的ACE活性有显著的抑制作用,从而使大鼠血清和肺组织中Ang II含量显著降低,而对正常血压的SD大鼠的血清和肺组织中ACE活性和Ang II含量影响不显著。     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from jellyfish Rhopilema esculentum

Two angiotensin I converting enzyme (ACE) inhibitory peptides were isolated from jellyfish Rhopilema esculentum protein hydrolysates prepared with pepsin and papain. Consecutive purification methods, including ion-exchange chromatography, size-exclusion chromatography and reverse-phase high-performance liquid chromatography, were used for isolation of ACE inhibitory peptides. The amino acid sequence of the peptides was identified as Gln-Pro-Gly-Pro-Thr and Gly-Asp-Ile-Gly-Tyr, respectively, and the IC₅₀ value of the purified peptides for ACE inhibitory activity was 80.67 μM and 32.56 μM. The purified peptides were evaluated for the antihypertensive effect in spontaneously hypertensive rats (SHR) after oral administration. Blood pressure decreased significantly after ingestion of the peptides. The results suggest that peptides derived from jellyfish R. esculentum may be beneficial as antihypertensive compounds in functional food resources.     

Identification of pro-drug type ACE inhibitory peptide sourced from porcine myosin B: Evaluation of its antihypertensive effects in vivo

This study aimed to identify a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from porcine skeletal myosin B. Proteins were hydrolyzed with pepsin and the hydrolysates were then subjected to various types of chromatography to isolate the active peptides. The 50% inhibitory concentrations of Lys–Arg–Val–Ile–Gln–Try (M6 a novel peptide) and Val–Lys–Ala–Gly–Phe (A5, a peptide discovered by Ukeda et al. (1991)) were 6.1 and 20.3 μM, respectively. As a result of a homology search, it was determined that the M6 peptide originated from myosin and peptide A5 was of actin origin. M6 is a novel ACE inhibitory peptide, whose activity was shown to be the strongest amongst the previously published myosin-originated peptides. Kinetic evaluations showed that both peptides are competitive inhibitors of ACE. Based on their activity against ACE, M6 was classified as a pro-drug conformer and A5 was classified as a substrate conformer. When both peptides were administered orally to spontaneously hypertensive rats at doses of 10 mg/kg, temporal hypertension was observed after 6 h. This study suggests that M6 and A5 are peptides that may serve several purposes. Based on their remarkable antihypertensive activity, we suggest that M6 and A5 may have potential applications as functional food, which could be used as nutraceutical compounds.     

Development of a salmon protein hydrolysate that lowers blood pressure

A salmon protein hydrolysate (SPH) was developed containing several angiotensin I-converting enzyme (ACE) inhibitory tripeptides the most abundant of which were Val-Leu-Trp, Val-Phe-Tyr, and Leu-Ala-Phe. Simulated digestion experiments showed that active constituents of SPH would survive in the digestive tract and be available for absorption into the bloodstream. In fact, ACE inhibitory activity was improved following simulated digestion suggesting that there were larger peptides in SPH that might contribute to bioactivity in vivo. A single oral dose (1,500 mg/kg body mass) of SPH significantly lowered blood pressure in spontaneously hypertensive rats (SHR). The treatment of SHR with either SPH fraction (<3,000 Da) or SPH fraction (>3,000 Da) reduced blood pressure. We conclude that the ability of SPH to lower blood pressure is due to a combination of ACE inhibitory tripeptides as identified, as well as additional unknown, peptide species that are generated during digestion of SPH in the gastrointestinal tract.     

Isolation and characterisation of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from the algae protein waste

A hendeca-peptide with angiotensin I-converting enzyme (ACE) inhibitory activity was isolated from the pepsin hydrolysate of algae protein waste, a mass-produced industrial by-product of an algae essence from microalgae, Chlorella vulgaris. Edman degradation revealed its amino acid sequence to be Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe. Inhibitory kinetics revealed a non-competitive binding mode with IC₅₀ value against ACE of 29.6 μM, suggesting a potent amount of ACE inhibitory activity compared with other peptides from the microalgae protein hydrolysates which have a reported range between 11.4 and 315.3 μM. In addition, the purified hendeca-peptide completely retained its ACE inhibitory activity at a pH range of 2–10, temperatures of 40–100 °C, as well as after treatments in vitro by a gastrointestinal enzyme, thus indicating its heat- and pH-stability. The combination of the biochemical properties of this isolated hendeca-peptide and a cheap algae protein resource make an attractive alternative for producing a high value product for blood pressure regulation as well as water and fluid balance.     

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).     

Antihypertensive mechanism of a peptide-enriched soy sauce-like seasoning: the active constituents and its suppressive effect on renin-angiotensin-aldosterone system

We previously reported that a peptide-enriched soy sauce-like seasoning called fermented soybean seasoning (FSS) demonstrated antihypertensive effects both in spontaneously hypertensive rats (SHR) and Dahl salt-sensitive rats. Angiotensin I-converting enzyme (ACE) inhibitory substances (9 kinds of dipeptides and a nicotianamine) were identified from FSS. In the present study, we clarified the mechanisms underlying the antihypertensive effects of FSS in SHR. FSS was divided into the nicotianamine fraction and the peptide fraction. The peptide fraction was found to exert a more prevalent antihypertensive effect than the nicotianamine fraction in SHR. Among the peptides, we identified Gly-Tyr and Ser-Tyr as the 2 primary substances in FSS that contributed to the antihypertensive effect in SHR. These peptides were neither degraded by acid nor gastrointestinal proteases, and were absorbed into the circulating blood. FSS (2000 mg/kg) exerted ACE inhibitory activity in the lung of rats and provided a decrease (P= 0.0067) in the level of serum aldosterone after a single oral administration in SHR, resulting in the antihypertensive effect. The antihypertensive mechanism was found to be similar to therapeutic ACE inhibitors and other food-derived ACE inhibitory peptides, which are in wide use and are recognized as safe.     

Purification and identification of a ACE inhibitory peptide from oyster proteins hydrolysate and the antihypertensive effect of hydrolysate in spontaneously hypertensive rats

Oyster (Crassostrea talienwhanensis Crosse) proteins were produced from fresh oyster and subsequently digested with pepsin. The separations were performed with a Sephadex LH-20 gel filtration chromatography and a RP-HPLC. A purified peptide with sequence Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (VVYPWTQRF) was firstly isolated and characterized from oyster protein hydrolysate and its ACE inhibitory activity was determined with IC₅₀ value of 66 μmol/L in vitro. Stability study for ACE inhibitory activity showed that the isolated nonapeptide had the good heat and pH stability and strong enzyme-resistant properties against gastrointestinal proteases. Kinetic experiments demonstrated that inhibitory kinetic mechanism of this peptide was non-competitive and its K_m and K_i values were calculated. The yield of this peptide from oyster proteins was 8.5%. Furthermore, the oyster protein hydrolysate (fraction II), prepared by pepsin treatment firstly exhibited antihypertensive activity when it was orally administered to spontaneously hypertensive rat (SHR) at a dose of 20 mg/kg. These results demonstrated that the hydrolysate from oyster proteins prepared by pepsin treatment could serve as a source of peptides with antihypertensive activity.     

Pilot-scale production of low molecular weight peptides from corn wet milling byproducts and the antihypertensive effects in vivo and in vitro

A pilot-scale production was developed to enhance the value of proteins from corn gluten meal (CGM). Corn protein isolate (CPI) with high protein content (90.68%) was obtained through heat treatment of CGM (150 kg) in aqueous alkaline solution. Two-step enzymatic hydrolysis and multistage separation were applied to enrich corn oligopeptides (COP) with low molecular weights, 96.77% of which were less than 1000 Da. The greatest antihypertensive effect of COP treatment in spontaneously hypertensive rats (SHR) was observed at a dose of 0.45 g/kg. One major ACE-inhibitory peptide, Ala-Tyr, was identified and quantified (9.16 ± 0.08 mg/g) from COP. The ACE inhibitory activity of Ala-Tyr (IC₅₀ = 0.037 mg/ml) was over 27 times higher than that of COP (IC₅₀ = 1.020 mg/ml). The results indicate that COP may be a source of natural antihypertensive compounds that could be used for drugs or functional food ingredients.