ACE-inhibitory Effect Research Articles

Identification, structure-activity relationship and in silico molecular docking analyses of five novel angiotensin I-converting enzyme (ACE)-inhibitory peptides from stone fish (Actinopyga lecanora) hydrolysates.

Stone fish is an under-utilized sea cucumber with many health benefits. Hydrolysates with strong ACE-inhibitory effects were generated from stone fish protein under the optimum conditions of hydrolysis using bromelain and fractionated based on hydrophobicity and isoelectric properties of the constituent peptides. Five novel peptide sequences with molecular weight (mw) < 1000 daltons (Da) were identified using LC-MS/MS. The peptides including Ala-Leu-Gly-Pro-Gln-Phe-Tyr (794.44 Da), Lys-Val-Pro-Pro-Lys-Ala (638.88 Da), Leu-Ala-Pro-Pro-Thr-Met (628.85 Da), Glu-Val-Leu-Ile-Gln (600.77 Da) and Glu-His-Pro-Val-Leu (593.74 Da) were evaluated for ACE-inhibitory activity and showed IC50 values of 0.012 mM, 0.980 mM, 1.310 mM, 1.440 mM and 1.680 mM, respectively. The ACE-inhibitory effects of the peptides were further verified using molecular docking study. The docking results demonstrated that the peptides exhibit their effect mainly via hydrogen and electrostatic bond interactions with ACE. These findings provide evidence about stone fish as a valuable source of raw materials for the manufacture of antihypertensive peptides that can be incorporated to enhance therapeutic relevance and commercial significance of formulated functional foods.

Plasma concentrations and ACE-inhibitory effects of tryptophan-containing peptides from whey protein hydrolysate in healthy volunteers

The tryptophan-containing dipeptides isoleucine-tryptophan (IW) and tryptophan-leucine (WL) are angiotensin-converting enzyme (ACE)-inhibitors in vitro. These peptides are released by enzymatic hydrolysis of bovine whey protein. To exhibit ACE inhibition in vivo, peptides need to be absorbed into the circulatory system. This study aimed to determine the in vivo ACE-inhibitory potency of a whey protein hydrolysate (MPH), containing IW and WL, and to quantify plasma concentrations of these peptides after oral administration of MPH in healthy volunteers. Additionally, changes in blood pressure were investigated. After intake of 5 and 50g MPH, plasma ACE activity was reduced to 86.4 ± 5.9 and 75.1 ± 6.9% of baseline activity, respectively. Although a clear ACE inhibition was measured, no effect on blood pressure was seen. Basal plasma concentrations of the tryptophan-containing dipeptides were 2.8 ± 0.7nM for IW and 10.1 ± 1.8nM for WL. After intake of 5-50g MPH, peptide concentrations were dose dependently elevated to values between 12.5 ± 8.4 and 99.1 ± 58.7nM for IW and 15.0 ± 4.3-34.9 ± 19.4nM for WL. Administration of intact whey protein showed a minor ACE inhibition, probably caused by release of inhibitory peptides during gastrointestinal digestion. The increase of WL in plasma after intake of intact protein was similar to that determined after intake of MPH. In contrast, resulting IW concentrations were much lower after intake of intact whey protein when compared to MPH administration. After intake of MPH, plasma ACE activity decreased in parallel to the increase of IW and WL plasma concentrations. However, the resulting peptide concentrations cannot fully explain the reduction of ACE activity in plasma with a direct enzyme inhibition. Therefore, this study points to a gap in the understanding of the inhibitory action of these peptides in vivo. Thus, to further develop innovative food additives with ACE activity diminishing capabilities, it appears mandatory to better characterize the mode of action of these peptides.

Biotechnological production of the angiotensin-converting enzyme inhibitory dipeptide isoleucine-tryptophan.

Peptides with angiotensin-converting enzyme (ACE)-inhibitory and antihypertensive effects are suggested as innovative food additives to prevent or treat hypertension. Currently, these substances are isolated from food proteins following nonselective hydrolysis as a mixture of ACE-inhibitory peptides and other protein fragments. This study presents an innovative biotechnological method, based on recombinant DNA technology that was established to specifically produce the ACE-inhibitory dipeptide isoleucine-tryptophan. In a first step, a repetitive isoleucine-tryptophan construct fused to the maltose-binding protein was generated and expressed in Escherichia coli BL21 cells. The chromatographically purified recombinant fusion protein was enzymatically hydrolyzed using α-chymotrypsin to liberate the dipeptide isoleucine-tryptophan. The identity of the liberated isoleucine-tryptophan was confirmed by MS and derivatization of its N-terminus. The ACE-inhibitory effect of the recombinant dipeptide on soluble and membrane bound ACE was found to be indistinguishable from the inhibitory potential of the chemically produced commercially available dipeptide. The established experimental strategy represents a promising approach to the biotechnical production of sufficient amounts of recombinant peptide-based ACE-inhibitory and antihypertensive substances that are applicable as functional food additives to delay or even prevent hypertension.

Abstract P488: Antihypertensive Effects of Lactotripeptides on Unattended Office and Abulatory Blood Pressure. A Randomized, Double-blind, Placebo-controlled Study

Milk fermentation by Lactobacillus helveticus produces some tripeptides (VPP and IVP) with sustained ACE-inhibitor activity that have shown to lower blood pressure (BP) in experimental animals and in humans. Grana Padano DOP, an Italian cheese, has shown a potent in vitro ACE-inhibitory effect due to its high concentration of such tripeptides. Present data refer to a randomized, double-blind, placebo controlled, study in which 30 mild-moderate hypertensive patients received a dietary integration with Grana Padano DOP. At base-line all patients had BP not on target (&gt;140 and/or 90 mmHg) after their usual treatment and received a dietary integration with Grana Padano (1 ounce per day) and placebo, in cross over fashion.BP was evaluated at baseline and at the end of the active and placebo treatment (2 months each) by Office and unattended Office BP (using the BpTRU, average of 5 consecutive BP readings) and by ambulatory BP monitoring (ABPM). Results: dietary integration with Grana Padano resulted in a significant decreased in systolic and diastolic office and unattended office BP (-6 mmHg for systolic and - 5 mmHg for diastolic BP). ABPM confirmed a sustained antihypertensive effect of such integration (see table). In conclusion, the diaily integration with 1 ounce of Grana Padano DOP cheese caused a significant decrease in systolic and diastolic BP pressure in hypertensive patients. No changes in BMI, total and HDL cholesterol, triglycerides, glucose, serum sodium and potassium levels were observed. The statistically significant drop in BP may be considered also clinically significant since the majority (67%) of the patients reached normal BP levels at the end of active treatment.

Purification of Mucuna pruriens (L) peptide fractions and evaluation of their ACE inhibitory effect

Abstract The ACE inhibitory potential of Mucuna pruriens (L) protein derivatives was evaluated. M. pruriens (L) protein concentrate was sequentially hydrolyzed using pepsin and pancreatin. Biological activity in the hydrolysate and its ultrafiltered fractions was evaluated and that with the highest biological activity was purified by gel filtration chromatography (GFC) and reverse phase high-performance liquid chromatography (RP-HPLC). The ACE inhibition manner and amino acid profile of the purified fractions with the highest biological potential was registered. The IC50 value for hydrolysate was 13.0270 μ g / mL and for the ultrafiltered fractions ranged from 0.0009 to 5.7519 μ g / mL . The five fractions (F1–F5) obtained by GFC had IC50 values in the range of 0.401–0.969 μ g / mL . The two fractions (F1-1 and F1-2) obtained by RP-HPLC from F1 had IC50 values of 16.162 and 14.542 μ g / mL , respectively with a competitive inhibition mechanism and a high hydrophobic amino acids distribution.

Antihypertensive peptides from animal products, marine organisms, and plants.

Bioactive peptides from food proteins exert beneficial effects on human health, such as angiotensin-converting enzyme (ACE) inhibition and antihypertensive activity. Several studies have reported that ACE-inhibitory peptides can come from animal products, marine organisms, and plants-derived by hydrolyzing enzymes such as pepsin, chymotrypsin, and trypsin-and microbial enzymes such as alcalase, thermolysin, flavourzyme, and proteinase K. Different ACE-inhibitory effects are closely related with different peptide sequences and molecular weights. Sequences of ACE-inhibitory peptides are composed of hydrophobic (proline) and aliphatic amino acids (isoleucine and leucine) at the N-terminus. As result of this review, we assume that low molecular weight peptides have a greater ACE inhibition because lower molecular weight peptides have a higher absorbency in the body. Therefore, the ACE-inhibitory effect is closely related with the degree of enzymatic hydrolysis and the composition of the peptide sequence.

Bioactivity of an antihypertensive peptide expressed in Chlamydomonas reinhardtii

In this study, we developed a transplastomic C. reinhardtii strain that accumulates anti-hypertensive peptides. Tandem repeats of VLPVP peptide were included. PCR analysis confirmed the presence of the transgene in the modified strains. After in vitro digestion of biomass of a recombinant C. reinhardtii strain the VLVPV peptide was identified and quantified by HPLC. The highest expression line produced 0.292mg of recombinant protein per mg of freeze-dried biomass. Intragastric administration of the genetically modified strain to spontaneous hypertensive rats at a dose of 30mg/kg of body weight of recombinant protein significantly reduced systolic blood pressure. At the same dose, the recombinant protein exerts an ACE-inhibitory effect. This is the first study that indicates the potential of this microalga producing an antihypertensive peptide as a dietary supplement for hypertension patients.

Angiotensin- I- converting enzyme (ACE) inhibitory peptides from Pacific cod skin gelatin using ultrafiltration membranes

Angiotensin- I- converting enzyme (ACE) is crucial in the control of hypertension and the development of type- 2 diabetes and other diseases associated with metabolic syndrome. The aim of this work was to utilize Pacific cod skin to purify ACE inhibitory peptides. First, gelatin was extracted from Pacific cod skin and hydrolyzed with several enzymes (pepsin, papain, α-chymotrypsin, trypsin, neutrase, and alcalase). The pepsin hydrolysate showed the strongest ACE inhibitory effect and was further fractionated into different ranges of molecular weight (<1, 1–5, 5–10, and >10kDa) using ultrafiltration (UF) membranes. The peptic hydrolysate below 1kDa resulted in two potent ACE inhibitory peptides, GASSGMPG (662Da) and LAYA (436Da), with IC50 values (concentration required to decrease the ACE activity by 50%) of 6.9 and 14.5μM, respectively. Moreover, to explore the interaction between the peptides and ACE molecule, the tertiary structure of ACE and docking simulation to the peptides were predicted using Docking Server. Pacific cod peptides can be used as functional food ingredients to prevent hypertension and its related diseases.

Antioxidant activity and ACE-inhibitory of Class II hydrophobin from wild strain Trichoderma reesei

There are several possible uses of the Class II hydrophobin HFBII in clinical applications. To fully understand and exploit this potential however, the antioxidant activity and ACE-inhibitory potential of this protein need to be better understood and have not been previously reported. In this study, the Class II hydrophobin HFBII was produced by the cultivation of wild type Trichoderma reesei. The crude hydrophobin extract obtained from the fermentation process was purified using reversed-phase liquid chromatography and the identity of the purified HFBII verified by MALDI-TOF (molecular weight: 7.2kDa). Subsequently the antioxidant activities of different concentrations of HFBII (0.01–0.40mg/mL) were determined. The results show that for HFBII concentrations of 0.04mg/mL and upwards the protein significantly reduced the presence of ABTS+ radicals in the medium, the IC50 value found to be 0.13mg/mL. Computational modeling highlighted the role of the amino acid residues located in the conserved and exposed hydrophobic patch on the surface of the HFBII molecule and the interactions with the aromatic rings of ABTS. The ACE-inhibitory effect of HFBII was found to occur from 0.5mg/mL and upwards, making the combination of HFBII with strong ACE-inhibitors attractive for use in the healthcare industry.

Acceptance of home blood pressure monitoring by patients with hypertension: results from the Heart Healthy Lenoir study

Cow-milk fermentation induced by Lactobacillus helveticus produces peptides with sustained ACE inhibitory activity, mainly dues to 2 tripeptides (valyl-prolyl-proline and isoleucil-prolyl-proline) that have shown to lower blood pressure (BP) in experimental animals and in humans. Grana Padano DOCG, an Italian semi-fat hard cheese, has shown a potent in-vitro ACE-inhibitory effect due its high concentration of such tripeptides. In a previous randomized, open-label, controlled study, the daily dietary integration with Grana Padano cheese was accompanied by significant decrease in systolic and diastolic BP in hypertensive patient (Crippa et al, J Clin Hypertens, 2013). Present data refers to a randomized, double-blind, placebo-controlled, cross-over study in which 30 mild-moderate hypertensive individuals, presenting with BP values not on target (>140 and /or >90 mmHg) after their usual and stable (3 months at least) treatment. All patients received, in random fashion, a dietary integration with Grana Padano Cheese (30 g per day) and placebo (obtained with flavored bread mixed with fats and salts in concentrations equal to those of the cheese). BP was evaluated at baseline and at the end of the active and placebo treatment (2 months each) by: -Office BP (OBP) -Automated Office BP -Ambulatory BP (ABP) monitoring. Automated Office BP was measured by using the BpTRU, (VSM MedTech Ltd, Vancouver, Canada), an automated oscillometric device that provides multiple blood pressure measurements in an office setting in the absence of a healthcare professional. It allows 6 consecutive readings at 1 minute intervals and provides the average of the last 5 measurements. Results: Dietary integration with Grana Padano cheese resulted in a significant decrease in systolic and diastolic Office and Automated Office BP (BpTRU) mean values (Student’s t test: p< 0.05 and p < 0.025). No changes in BMI, total and HDL cholesterol, triglycerides, blood glucose, serum sodium and potassium levels were found after the dietary integration both with respect to baseline and placebo.

Randomized, double-blind, placebo-controlled, cross-over study on the antihypertensive effect of dietary integration with Grana Padano DOCG cheese

Cow-milk fermentation induced by Lactobacillus helveticus produces peptides with sustained ACE inhibitory activity, mainly dues to 2 tripeptides (valyl-prolyl-proline and isoleucil-prolyl-proline) that have shown to lower blood pressure (BP) in experimental animals and in humans. Grana Padano DOCG, an Italian semi-fat hard cheese, has shown a potent in-vitro ACE-inhibitory effect due its high concentration of such tripeptides. In a previous randomized, open-label, controlled study, the daily dietary integration with Grana Padano cheese was accompanied by significant decrease in systolic and diastolic BP in hypertensive patient (Crippa et al, J Clin Hypertens, 2013). Present data refers to a randomized, double-blind, placebo-controlled, cross-over study in which 30 mild-moderate hypertensive individuals, presenting with BP values not on target (>140 and /or >90 mmHg) after their usual and stable (3 months at least) treatment. All patients received, in random fashion, a dietary integration with Grana Padano Cheese (30 g per day) and placebo (obtained with flavored bread mixed with fats and salts in concentrations equal to those of the cheese). BP was evaluated at baseline and at the end of the active and placebo treatment (2 months each) by: -Office BP (OBP) -Automated Office BP -Ambulatory BP (ABP) monitoring. Automated Office BP was measured by using the BpTRU, (VSM MedTech Ltd, Vancouver, Canada), an automated oscillometric device that provides multiple blood pressure measurements in an office setting in the absence of a healthcare professional. It allows 6 consecutive readings at 1 minute intervals and provides the average of the last 5 measurements. Results: Dietary integration with Grana Padano cheese resulted in a significant decrease in systolic and diastolic Office and Automated Office BP (BpTRU) mean values (Student’s t test: p< 0.05 and p < 0.025). No changes in BMI, total and HDL cholesterol, triglycerides, blood glucose, serum sodium and potassium levels were found after the dietary integration both with respect to baseline and placebo.

Inhibitory effect of leaves extracts of Ocimum basilicum and Ocimum gratissimum on two key enzymes involved in obesity and hypertension in vitro.

Aim:To evaluate the phenolics composition and inhibitory effect of the leaves extracts of Ocimum basilicum and Ocimum gratissimum on two key enzymes (pancreatic lipase [PL] and angiotensin 1-converting enzyme [ACE]) involved in obesity and hypertension in vitro.Materials and Methods:The phenolics (flavonoids and phenolic acids) were quantified using high-performance liquid chromatography coupled with diode array detection. PL and ACE inhibitory effects; DPPH* and ABTS*+ scavenging activities of the extracts were tested using spectrophotometric methods.Results:O. basilicum had the following major phenolics: Rutin, quercetin, and quercitrin (flavonoids); caffeic, chlorogenic, and gallic acids (phenolic acids); while O. gratissimum had the following major phenolics: Rutin, quercitrin, and luteolin (flavonoids); ellagic and chlorogenic acids (phenolic acids). “Extracts of both plants inhibited PL and ACE; scavenged DPPH* in a dose-dependent manner”. O. gratissimum extract was more potent in inhibiting PL (IC50: 20.69 µg/mL) and ACE (IC50: 29.44 µg/mL) than O. basilicum (IC50: 52.14 µg/mL and IC50: 64.99 µg/mL, against PL and ACE, respectively). O. gratissimum also scavenged DPPH* and ABTS*+ more than O. basilicum.Conclusion:O. basilicum and O. gratissimum leaves could be used as functional foods for the management of obesity and obesity-related hypertension. However, O. gratissimum may be more effective than O. basilicum.

In vitro ACE-inhibitory peptide KGYGGVSLPEW facilitates noradrenaline release from sympathetic nerve terminals: Relationship with the lack of antihypertensive effect on spontaneous hypertensive rats

This study aimed to validate the antihypertensive activity of the angiotensin-converting enzyme (ACE)-inhibitor whey protein hydrolysate (WPH) obtained through the action of proteolytic enzymes from Cynara Cardunculus. The antihypertensive activity of WPH fractions containing peptides with molecular weight below 3kDa (Whey<3kDa) and 1kDa (Whey<1kDa) along with the antihypertensive activity of three potent ACE-inhibitory peptide sequences (DKVGINYW, DAQSAPLRVY and KGYGGVSLPEW), previously identified in WPH, were also investigated. In parallel, the influence of KGYGGVSLPEW (the most potent ACE-inhibitory peptide sequence) on AT1 receptors (a common pharmacological target of antihypertensive therapies beyond ACE), was evaluated. The effect of WPH and fractions (300mg/kg) and peptide sequences (5mg/kg) on systolic, diastolic and mean blood pressure was evaluated by telemetry on spontaneously hypertensive rats (SHR), after single oral administration. Despite their ACE-inhibitory effect in vitro, neither WPH, Whey <3kDa, Whey <1kDa or peptide sequences exhibited antihypertensive activity. In addition, KGYGGVSLPEW was not only devoid of AT1 receptor antagonism but, on the contrary, had a similar effect to that of Ang II by facilitating the noradrenaline release from sympathetic nerve terminals. In vitro ACE blockade does not always correlate with antihypertensive activity and food-derived peptides cannot be classified as antihypertensive agents based exclusively on in vitro assays. The absence of an antihypertensive effect may also be a result of the interaction of these compounds with other components of the systems involved in the blood pressure control.

Enzymatic modification of sesame seed protein, sourced from waste resource for nutraceutical application

The functional characteristics which include protein solubility at different pH, emulsifying and foaming properties, degree of hydrolysis, molecular weight distribution, antioxidant and ACE inhibitory activity of sesame protein hydrolysates prepared with pepsin, papain and alcalase enzymes were evaluated. The rate of degree of hydrolysis was found to reach maximum (25–30%) within the first time fragment i.e 10min but 80% of hydrolysis was obtained in 120min with alcalase. SDS-PAGE of hydrolysates with papain, pepsin and alcalase evinced bands of low molecular weight protein of 14.3kDa and even lower for alcalase treatment of 120min. Hydrolysates so formed were of improved functional properties as evident from emulsifying and foaming property. Hydrolysis with different proteases enhanced the protein solubility significantly at pH 7.0. Antioxidative assay revealed radical scavenging activity of the hydrolysates with papain hydrolysates showing maximum antioxidative efficacy. The ultra-filtered peptide fractions which showed comparable ACE inhibitory activity were sequenced by MALDI-TOF and matched to that of previously identified ACE inhibitory peptides. The results corroborate the ACE inhibitory effect of the peptides. Hence, these highly bioactive protein hydrolysates produced from waste sesame meals can be successfully employed in various functional food formulations.

Isolation of angiotensin I-converting enzyme inhibitor from pepsin hydrolysate of porcine hemoglobin

Angiotensin I-converting enzyme (ACE) inhibitors have been widely used as antihypertensive agents. However, most synthetic ACE inhibitors ineluctably have severe side effects. Researchers have focused on various ACE-inhibitory peptides derived from dietary food. In the present study, we reported peptides produced from porcine blood, an important food in Asian countries. Through enzymatic hydrolysis, we found that peptides from this animal compound have ACE-inhibitory effects. Porcine hemoglobin was hydrolyzed using ordinary proteases, including alcalase, trypsin, neutral, papain, protamex, and pepsin. Results showed that pepsin was the most efficient protease in producing active peptides, and the pepsin hydrolysate of porcine hemoglobin showed the highest activity (IC50 = 1.53 ± 0.03 mg/mL). Combining DA 201-C macroporous resin chromatography, Sephadex LH-20 gel chromatography, and reversed-phase high-performance liquid chromatography, the fraction 2-IV was purified from pepsin hydrolysis of porcine hemoglobin; this compound exhibited the highest ACE-inhibitory activity (IC50 = 0.02 ± 0.01 mg/mL). Through Edman degradation, we also found that the exact amino acid sequence of fraction 2-IV was Gln–Glu–Leu–Pro–Gly. The results indicated that porcine hemoglobin peptides possessed significant ACE-inhibitory effect in vitro, which is an important complement of the previous work.

ACE-inhibitory Effect and Physicochemical Characteristics of Yogurt Beverage Fortified with Whey Protein Hydrolysates

This study investigated the ACE-inhibitory effect of yogurt beverage fortified with hydrolysates as well as the suitabilityof hydrolysates as a nutraceutical additive to yogurt beverage. Three whey protein hydrolysates hydrolyzed by alcalase,protamex, and trypsin were each added to yogurt beverage at concentrations of 1.25, 2.5, and 5 mg/mL. Yogurt beveragefortified with 2.5 mg/mL of hydrolysates had 61-69% ACE-inhibitory activity, whereas yogurt beverage fortified with 5 mg/mL of hydrolysates showed 74% ACE-inhibitory activity. There were no significant differences in ACE-inhibitory activitybetween the alcalase or protamex hydrolysates during storage; however, trypsin hydrolysate exhibited significant differ-ences. On the other hand, physicochemical characteristics such as pH (3.47-3.77), titratable acidity (0.81-0.84%), colority,viable cell count, and sensory qualities were not significantly different among the tested yogurt beverage samples duringstorage. These results showed that yogurt beverage fortified with whey protein hydrolysates maintained antihypertensiveactivity and underwent no unfavorable changes in physicochemical characteristics regardless of enzyme type.Key words: angiotensin-converting enzyme, whey protein hydrolysate, yogurt beverage

Novel whey-derived peptides with inhibitory effect against angiotensin-converting enzyme: In vitro effect and stability to gastrointestinal enzymes

Whey protein concentrate (WPC) was subjected to enzymatic hydrolysis by proteases from the flowers of Cynara cardunculus, and the resulting angiotensin-converting enzyme (ACE)-inhibitory effect was monitored. The whole WPC hydrolysate exhibited an IC 50 value of 52.9 ± 2.9 μg/mL, whereas the associated peptide fraction with molecular weight below 3 kDa scored 23.6 ± 1.1 μg/mL. The latter fraction was submitted to RP-HPLC, and 6 fractions were resolved that exhibited ACE-inhibitory effects. Among the various peptides found, a total of 14 were identified via sequencing with an ion-trap mass spectrometer. Eleven of these peptides were synthesized de novo – to validate their ACE-inhibitory effect, and also to ascertain their stability when exposed to simulated gastrointestinal digestion. Among them, three novel, highly potent peptides were found, corresponding to α-lactalbumin f(16–26) – with the sequence KGYGGVSLPEW, α-lactalbumin f(97–104) with DKVGINYW, and β-lactoglobulin f(33–42) with DAQSAPLRVY; their IC 50 values were as low as 0.80 ± 0.1, 25.2 ± 1.0 and 13.0 ± 1.0 μg/mL, respectively. None of them remained stable in the presence of gastrointestinal enzymes: they were partially, or even totally hydrolyzed to smaller peptides – yet the observed ACE-inhibitory effects were not severely affected for two of those peptides.

ACE inhibitory and hydrolytic enzyme activities in textured vegetable protein in relation to the solid state fermentation period using Bacillus subtilis HA

Textured vegetable protein (TVP) was fermented by solid-state fermentation using Bacillus subtilis HA and production of biologically active compounds were examined with fermentation time. The TVP fermented resulted in the highest protease activity at 48 hr, where as α-amylase activity was the highest at 24 hr. During the fermentation the peptide and tyrosine content increases drastically and then it decrease gradually after 48 hr. The fast protein transition to low molecular peptides in early fermentation period was confirmed with sodium dedecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) analysis. Fibrinolytic enzyme activity proportionately increased with the fermentation time and the highest enzyme activity was 25.2 unit/g after fermentation for 120 hr. In the evaluation of ACE inhibitory effect, IC50 value of fermented TVP and its crude peptides was similar with 2.19 and 1.21 mg/mL, respectively. Peptide fraction below 3,000 Da showed 0.5 mg/mL of IC50 value. The peptide fraction (III) with molecular weight of 500–999 Da separated from SEC was consisted of 7 amino acids, indicated 0.094 mg/mL of IC50 value which is 23 times higher than the fermented TVP.

The antihypertensive effect of fermented milk in individuals with prehypertension or borderline hypertension

Fermented milk (FM) with putative antihypertensive effect in humans could be an easy applicable lifestyle intervention against hypertension. The mode of action is supposed to be through active milk peptides, shown to possess in vitro ACE-inhibitory effect. Blood pressure (BP) reductions upto 23 mm Hg have been reported in spontaneously hypertensive rats fed FM. Results from human studies of the antihypertensive effect are inconsistent. However, many studies suffer from methodological weaknesses, as insufficient blinding and the use of office BP measurements. We conducted a randomised, double-blind placebo-controlled study of the antihypertensive effect of Lactobacillus helveticus FM in 94 prehypertensive and borderline hypertensive subjects. The participants were randomised into three treatment groups with a daily intake of 150 ml of FM, 300 ml of FM or placebo (chemically acidified milk). The primary outcome was repeated 24-h ambulatory BP measurements. There were no statistically significant differences in the outcome between the groups (systolic BP (SBP), P=0.9; diastolic BP (DBP), P=0.2). However, the group receiving 300 ml FM had reduced BP across the 8-week period in several readings, which could be compatible with a minor antihypertensive effect. Heart rate and lipids remained unchanged between groups. Hence, our study does not support earlier studies measuring office BP-measurements, reporting antihypertensive effect of FM. Based on straight performed 24-h ambulatory BP measurements, milk fermented with Lactobacillus helveticus does not posses significant antihypertensive effect.

Macroporous resin purification of grass carp fish (Ctenopharyngodon idella) scale peptides with in vitro angiotensin-I converting enzyme (ACE) inhibitory ability

The adsorption dynamics and thermodynamics of grass carp fish scale peptides (FSPs) onto non-polar macroporous resins (MARs), DA201-C, have been investigated. The adsorption of FSPs was affected by time, pH and peptide concentration. The adsorption process followed the Langmuir adsorption isotherm, and was endothermic (ΔH <43kJ/mol). The predominant force in adsorption of FSPs onto DA201-C was hydrophobic. Depending on this force, the dynamic adsorption and gradient desorption results showed that DA201-C resins were good at desalting and enriching peptides with higher contents of hydrophobic amino acids, and these peptides had higher ACE inhibitory capabilities in vitro. The lowest concentration at which the eluted fraction possessed half of its original ACE activity (IC₅₀) was 0.13mg/ml. The results indicated that fish scale peptides produced showed good ACE-inhibitory effect in vitro and fish scales are a good source of peptides with in vitro ACE inhibitory activity.