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This study investigates the characterization, mechanisms of action, structure-activity relationships, and in vivo antihypertensive effects of ACE inhibitory peptides derived from sufu hydrolysate following simulated gastrointestinal digestion. Sufu was enzymatically digested using pepsin, trypsin, and chymotrypsin to mimic gastrointestinal conditions, followed by ultrafiltration to fractionate the peptides based on molecular weight. The fraction under 1 kDa exhibited the highest ACE inhibitory activity. LC-MS/MS analysis identified 119 peptide fragments, with bioinformatics screening highlighting 41 peptides with potential ACE inhibitory properties. Among these, two peptides, AWR and LLR, were selected and synthesized for in vitro validation, displaying IC 50 values of 98.04 ± 2.56 μM and 94.01 ± 5.07 μM, respectively. Stability tests showed that both peptides maintained their ACE inhibitory activity across various temperatures and pH levels. Molecular docking and Highest Occupied Molecular Orbital analysis indicated strong binding interactions between these peptides and ACE, with the second-position tryptophan in AWR and the N-terminal leucine in LLR identified as key bioactive sites. These findings were further supported by molecular dynamics simulations, which confirmed the stability of the peptide-ACE complexes. In vivo studies using spontaneously hypertensive rats demonstrated significant reductions in both systolic and diastolic blood pressure, indicating that AWR and LLR have strong antihypertensive potential. This study illustrates that ultrafiltration, combined with LC-MS/MS and bioinformatics analysis, is an effective approach for the rapid screening of ACE inhibitory peptides. These results not only enhance our understanding of sufu-derived peptides but also offer promising implications for hypertension management.

The stability of bioactive peptides under various food processing conditions is the basis for their use in industrial manufacturing. This study aimed to identify natural ACE inhibitors with excellent stability and investigate their physicochemical properties and putative molecular mechanisms. Five novel ACE inhibitory peptides (QDPLFPL, FPGVSPF, SPAQLLPF, LVPYRP, and WYWPQ) were isolated and identified using RP-HPLC and Nano LC-MS/MS with foxtail millet protein hydrolysates as the raw material. These peptides are non-toxic and exhibit strong ACE inhibitory activity in vitro (IC 50 values between 0.13 mg mL -1 and 0.56 mg mL -1 ). In addition to QDPLFPL, FPGVSPF, SPAQLLPF, LVPYRP, and WYWPQ have excellent human intestinal absorption. Compared to FPGVSPF and SPAQLLPF, the stable helical structure of LVPYRP and WYWPQ allows them to maintain high stability under conditions that mimic gastrointestinal digestion and various food processing (temperatures, pH, sucrose, NaCl, citric acid, sodium benzoate, Cu 2+ , Zn 2+ , K + , Mg 2+ , Ca 2+ ). The results of molecular docking and molecular dynamics simulation suggest that LVPYRP has greater stability and binding capacity to ACE than WYWPQ. LVPYRP might attach to the active pockets (S1, S2, and S1') of ACE via hydrogen bonds and hydrophobic interactions, then compete with Zn 2+ in ACE to demonstrate its ACE inhibitory activity. The binding of LVPYRP to ACE enhances the rearrangement of ACE's active structural domains, with electrostatic and polar solvation energy contributing the most energy to the binding. Our findings suggested that LVPYRP derived from foxtail millet protein hydrolysates has the potential to be incorporated into functional foods to provide antihypertensive benefits.

Bioactive peptides derived from food are promising health-promoting ingredients that can be used in functional foods and nutraceutical formulations. In addition to the potency towards the selected therapeutic target, the bioavailability of bioactive peptides is a major factor regarding clinical efficacy. We have previously shown that a low molecular weight peptide fraction (LMWPF) from poultry by-product hydrolysates possesses angiotensin-1-converting enzyme (ACE-1) and dipeptidyl-peptidase 4 (DPP4) inhibitory activities. The present study aimed to investigate the bioavailability of the bioactive peptides in the LMWPF. Prior to the investigation of bioavailability, a dipeptide YA was identified from this fraction as a dual inhibitor of ACE-1 and DPP4. Gastrointestinal (GI) stability and intestinal absorption of the bioactive peptides ( i.e. , YA as well as two previously reported bioactive dipeptides (VL and IY)) in the LMWPF were evaluated using the INFOGEST static in vitro digestion model and intestinal Caco-2 cell monolayer, respectively. Analysis of peptides after in vitro digestion confirmed that the dipeptides were resistant to the simulated GI conditions. After 4 hours of incubation, the concentration of the peptide from the apical side of the Caco-2 cell monolayer showed a significant decrease. However, the corresponding absorbed peptides were not detected on the basolateral side, suggesting that the peptides were not transported across the intestinal monolayer but rather taken up or metabolized by the Caco2 cells. Furthermore, when analyzing the gene expression of the Caco-2 cells upon peptide stimulation, a down-regulation of peptide transporters, the transcription factor CDX2 , and the tight junction protein-1 ( TJP1 ) was observed, suggesting the specific effects of the peptides on the Caco-2 cells. The study demonstrated that bioactive dipeptides found in the LMWPF were stable through in vitro GI digestion; however, the overall bioavailability may be hindered by inadequate uptake across the intestinal barrier.

Studies have confirmed that yogurt has the activity of regulating blood pressure because it is rich in probiotic-fermented food-derived active peptides. There are also studies on angiotensin-converting enzyme inhibition (ACEI) peptide milk, but the bioactive molecules in it are still unclear. Therefore, in this study, we developed a peanut yogurt with ACEI activity, analyzed 1877 differential peptides and their antihypertensive pathways before and after fermentation using peptidomics, and identified three peptides (FLPYPY, QPPPSPPPFL and APFPEVFGK) with potential antihypertensive activity using molecular docking and chemical synthesis techniques. These results first elucidated the relationship between peanut yogurt peptides and antihypertensive function, demonstrated the benefits of peanut yogurt, and provided a theoretical basis for the application of probiotic fermented plant yogurt in health care.

The considerable value of whey is evident from its significant potential applications and contributions to the functional food and nutraceutical market. The by-products were individually obtained during functional chhurpi and novel soy chhurpi cheese production using defined lactic acid bacterial strains of Sikkim Himalaya's traditional chhurpi . Hydrolysis of substrate proteins by starter proteinases resulted in a comparable peptide content in whey and soy whey which was associated with antioxidant and ACE inhibition potential. Peptidome analysis of Lactobacillus delbrueckii WS4 whey and soy whey revealed the presence of several bioactive peptides including the multifunctional peptides PVVVPPFLQPE and YQEPVLGPVRGPFPIIV. In silico analyses predicted the antihypertensive potential of whey and soy whey peptides with strong binding affinity for ACE active sites. QSAR models predicted the highest ACE inhibition potential (IC 50 ) for the β-casein-derived decapeptide PVRGPFPIIV (0.95 μM) and the Kunitz trypsin inhibitor protein-derived nonapeptide KNKPLVVQF (16.64 μM). Chhurpi whey and soy whey can be explored as a valuable source of diverse and novel bioactive peptides for applications in designer functional foods development.

The salty oligopeptides from Stropharia rugosoannulata have been proven to be potential ACE inhibitors. To investigate the ACE receptor binding properties and interaction mechanisms of salty oligopeptides, the molecular interaction, dynamics simulation, and antihypertensive evaluation cross-validation strategy were employed to reveal the oligopeptides' binding reactions and modes with the ACE receptor. Single oligopeptide (ESPERPFL, KSWDDFFTR) had exothermic and specific binding reactions with the ACE receptor, driven by hydrogen bonds and van der Waals forces. The coexistence of the multiple oligopeptide molecules did not produce the apparent ACE receptor competition binding reactions. The molecular dynamics simulation verified that the two oligopeptides disturbed the ACE receptor's different residue regions. Both oligopeptides could form stable complexes with the ACE receptor. Based on the classification of 50 oligopeptides' binding modes, ESPERPFL and KSWDDFFTR belonged to different classes, and their receptor binding modes and sites complemented, resulting in a potential synergistic effect on ACE inhibition. The antihypertensive effect of KSWDDFFTR and its distribution in the body were evaluated using SHR rats orally and ICR mice by tail vein injection, and KSWDDFFTR had antihypertensive effects within 8 h. The study provides a theoretical basis for understanding salty oligopeptides' ACE receptor binding mechanism and their antihypertensive effects.

In this study, the effects of Lactiplantibacillus plantarum M11 ( Lb. plantarum M11) in conjunction with sodium caseinate on the characteristics and angiotensin converting enzyme (ACE) inhibitory activity of yogurt were investigated. ACE inhibitory peptides (ACEIPs) in yogurt were identified by nano-LC-MS/MS and potential ACEIPs were predicted by in silico and molecular docking methods. The results showed that the ACE-inhibitory activity of yogurt was significantly enhanced ( p < 0.05), while maintaining the quality characteristics of the yogurt. Thirteen ACEIPs in the improved yogurt (883 + M11-CS group) were identified, which were more abundant than the other yogurt groups (control 883 group, 883 + M11 group and 883-CS group). Two novel peptides with potential ACE inhibitory activity, YPFPGPIH and NILRFF, were screened. The two peptides showed PeptideRanker scores above 0.8, small molecular weight and strong hydrophobicity, and were non-toxic after prediction. Molecular docking results showed that binding energies with ACE were -9.4 kcal mol -1 and -10.7 kcal mol -1 , respectively, and could bind to the active site of ACE. These results indicated that yogurt with Lb. plantarum M11 and sodium caseinate has the potential to be utilized as a functional food with antihypertensive properties. The combination of ACEIP-producing strains and casein fortification could be an effective method to promote the release of ACEIPs from yogurt.

Peptides in milk fermented with Lactobacillus delbrueckii QS306 before and after ultrahigh pressure treatment were identified using proteomics. Subsequently, 16 stable tripeptides were screened out based on activity score prediction, PeptideCutter analysis, and hydrophobicity calculations. Among them, WRP, WSR, and YRP showed the best angiotensin-converting enzyme (ACE) inhibitory activity, and their semi-inhibitory concentrations were 46.707, 300.121, and 89.555 μM, respectively. WRP and WSR were competitive inhibitors, whereas YRP was non-competitive. Gastrointestinal simulation revealed that WRP and YRP had better gastrointestinal stability. The values of RMSD, Δ G bind , Δ G pol , and RSMF obtained from molecular dynamics simulation indicated that the interaction of WRP and ACE was stable. Thus, Lactobacillus delbrueckii QS306-fermented milk can serve as an important source of ACE inhibitory peptides both before and after ultrahigh pressure treatment. The strategy of in silico screening, activity evaluation, and molecular dynamics simulation adopted in this study can be applied to the large-scale screening of novel peptides with high ACE inhibitory activity.

Turtle shell as a food residue of Pelodiscus sinensis (a type of edible aquatic animal) is widely used in Traditional Chinese Medicine for hepatic fibrosis therapy. Previous studies have demonstrated that the peptides (<6 kDa) derived from turtle shells are considered effective components. The protein of turtle shells has important potential as a source of bioactive peptides which may play a role as ingredients in functional foods. In the present study, the protein of turtle shell was hydrolyzed using a two-enzyme combination. It was found that the hydrolysates obtained by a combination of pepsin and trypsin showed the highest anti-liver fibrosis activity relative to other combinations in a cell viability assay. The hydrolysates were separated and purified by ultra-filtration (<6 kDa), gel filtration chromatography (GFC) and high-performance liquid chromatography (HPLC). Subsequently, the sequences of purified peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Molecular docking was used to analyze the interaction of these peptides with the transforming growth factor-β 1 (TGF-β 1 ) receptor. Two (GPPGVPGPGPL, TSLPVPAPV) of these novel peptides displayed lower binding energies to the TGF-β 1 receptor (-8.18 kcal mol -1 , -8 kcal mol -1 ). Finally, the above two peptides were synthesized chemically and their in vitro anti-liver fibrosis activity was verified by MTT assay. Among them, GPPGVPGPGPL showed a better in vitro anti-liver fibrosis activity (IC50: 80.13 μM). We established a method to obtain anti-liver fibrosis peptides from turtle shells by using bioactivity-guided isolation with molecular docking. Turtle shell protein is an excellent source of anti-liver fibrosis peptides which can offer therapeutic and commercial benefits as an ingredient in functional foods.

The side effects of traditional antihypertensive drugs have driven people's interest in the discovery of novel angiotensin-I converting enzyme (ACE) inhibitory peptides with efficiency and safety. Spirulina possesses abundant proteins and is considered as a good source of bioactive peptides. To identify ACE inhibitory peptides, Spirulina protein was hydrolyzed by protease K, and the hydrolysate was separated by gel permeation chromatography and reverse phase HPLC. Thirty peptide sequences were identified from the ACE inhibitory fractions by LC-MS/MS, and 15 potential ACE inhibitory peptides were further selected by combined virtual screening and experimental investigation. Among the identified Spirulina peptides, TVLYEH (SpH-6, IC 50 = 2.88 μM) and LQAGGLF (SpH-7, IC 50 = 66.83 μM) were found to possess potent ACE inhibitory activity. The inhibition patterns of SpH-6 and SpH-7 were characterized as competitive inhibition. Molecular docking indicated that SpH-6 and SpH-7 could bind to the active pockets of ACE by forming hydrogen bonds, salt bridges and pi-pi stacking. Both SpH-6 and SpH-7 exhibited stable ACE inhibitory activity under different pH (2, 4, 6, 8, 10, 12) and temperature (0, 20, 40, 60, 80, 100 °C) conditions. The ACE inhibitory activity of SpH-6 decreased slightly after simulated gastrointestinal digestion, and SpH-7 was unstable against digestive enzymes. The in vivo antihypertensive effect demonstrated that oral administration of SpH-6 significantly reduced the systolic blood pressure and diastolic blood pressure of spontaneously hypertensive rats. The present study revealed the potential antihypertensive effects of SpH-6 and indicated that SpH-6 and Spirulina protein hydrolysate could be applied as nutritional supplements for blood pressure control.

In the study, seventeen angiotensin converting enzyme (ACE) inhibitory peptides were isolated from the protein hydrolysate of blue mussel ( Mytilus edulis ) and identified as MFR, MFV, FV, KP, QP, QVK, IK, YKV, IRK, MLKV, NFRPQ, YEGDP, WF, GPE, SWISS, SVEWK, and FKWH, respectively. Among them, IK, YEGDP, WF, and SWISS showed the strongest ACE inhibitory activity with IC 50 values of 0.77 ± 0.020, 0.19 ± 0.010, 0.40 ± 0.015, and 0.32 ± 0.017 mg mL -1 , respectively. Molecular docking study indicated that IK, YEGDP, WF, and SWISS exhibited better inhibitory activity attributed to its effective interaction with the active site of ACE by hydrogen bonding, electrostatic force and hydrophobic interaction. Furthermore, IK, YEGDP and WF perform an important protective function on human umbilical vein endothelial cells (HUVECs) by increasing nitric oxide (NO) content, decreasing endothelin-1 (ET-1) secretion, and antagonizing the adverse impact of norepinephrine on the secretion of NO and ET-1. In addition, YEGDP and WF could provide protection to HUVECs against H 2 O 2 damage by increasing superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and NO levels to decrease the contents of reactive oxygen species (ROS) and malondialdehyde. Therefore, seventeen ACE inhibitory peptides, especially YEGDP and WF, might be used as natural ingredients for the development of products with antihypertensive functions.

VGINYW is a highly active angiotensin I-converting enzyme (ACE) inhibitory peptide discovered from α-lactalbumin by an in vitro - in silico high throughput screening strategy. The aim of this study was to evaluate the antihypertensive effect of the peptide and the α-lactalbumin hydrolysates under 3 kDa (LH-3k), and illustrate the possible mechanism in spontaneously hypertensive rats (SHRs). SHRs were administered with VGINYW and LH-3k at doses of 5 mg per kg BW and 100 mg per kg BW, respectively. VGINYW and LH-3k could markedly decrease the systolic blood pressure (SBP) of the SHRs, and the maximal drops of 21 mmHg (2 h after administration) and 17 mmHg (4 h after administration) were achieved during the 8 hour test, respectively. When the agents were given once per day for 4 weeks, they caused a long-term decrease of 16 mmHg of SBP. VGINYW and LH-3k control the blood pressure through regulating the renin-angiotensin system by inhibiting the ACE activity and diminishing the angiotensin II level, and further upregulating the expression levels of the angiotensin-converting enzyme 2 and angiotensin type 2 receptor, and downregulating the expression of the angiotensin type 1 receptor. VGINYW and LH-3k could notably ameliorate the oxidative stress in the SHR as well. It is more important that the gavage of VGINYW and LH-3k could alleviate hypertension-associated intestinal microbiota dysbiosis by recovering the diversity of the gut microbiota and altering the key floras which are short chain fatty acid producers. In conclusion, VGINYW and LH-3k are effective functional ingredients for blood pressure control.

Bioactive peptides exhibiting angiotensin-converting enzyme (ACE) inhibitory effects and extracted from natural foods have potential as healthy and safe therapeutics for high blood pressure. The aim of this study was to isolate and purify ACE inhibitory peptides from rabbit meat protein hydrolysate, to explore the underlying mechanisms by molecular docking, and to evaluate the antihypertensive effects in vivo . A novel ACE inhibitory tetrapeptide Trp-Gly-Ala-Pro (WGAP) was identified and purified from a bromelain hydrolysate. WGAP acted against ACE in a non-competitive manner with an IC 50 of 140.70 ± 4.51 μM. It was resistant to enzymatic degradation by pepsin and trypsin in vitro . Molecular docking analysis indicated that WGAP formed stable hydrogen bonds with ACE residues His353, Ala354 and ALA356. In vivo , 100 mg kg -1 WGAP significantly reduced systolic and diastolic blood pressure in hypertensive rats by up to 42.66 ± 2.87 and 28.56 ± 2.71 mmHg, respectively, 4 h after oral administration. ACE inhibitory peptides derived from rabbit meat have potential antihypertensive effects and provide a new route for the exploration of novel hypertension inhibitors and the utilization of rabbit meat.

Pallenis spinosa is a medicinal plant which is used in folk medicine as curative or preventive remedies for various diseases. Individual phenolic compounds from the methanolic extracts of its flowers, leaves and stem were determined by the high performance liquid chromatography method (HPLC) and total phenolic contents (TPC) were evaluated by Folin-Ciocalteu assay. The stability and bioactivity (antioxidant activity, micellar cholesterol solubility, α-amylase, and angiotensin converting enzymes (ACE) inhibitory effects) of these extracts in the gastrointestinal environment was determined before and after their protection in hydroxypropylmethylcellulose (HPMC) capsules. HPLC analysis revealed the presence of thirteen phenolic compounds with nine flavonoids and four phenolic acids. Except for kaempferol, the twelve other compounds have not been previously detected in the aerial part of the studied plant. Quantification of phenolics by HPLC and Folin Ciocalteu methods revealed that the highest TPC was detected in the flower extracts (104.31 ± 0.80 and 145.73 ± 0.48 mg EGA per g of extract, respectively). Leaf extracts displayed the best antioxidant capacity against the two tested radicals DPPH and ABTS (IC50 = 1.24 ± 0.03 and 0.94 ± 0.02 mg mL-1, respectively), FRAP assay (IC50 = 0.50 ± 0.02 mg mL-1), α-amylase inhibitory (IC50 = 1.25 ± 0.00 mg mL-1) and angiotensin activity with an inhibitory percent of 30.10 ± 0.12%. The best activity shown by stem extracts was against micellar cholesterol solubility (67.57 ± 0.00%). A strong decrease in TPC and their bioactivity was observed after the gastrointestinal digestion (GID) in non encapsulated extracts. These results showed that P. spinosa is a good source of phenolic compounds and GID affects significantly their composition, content and bioactivity.

The oil processing industry generates significant quantities of lemon basil seed residue which is not currently used to any significant extent. However, this by-product has important potential as a source of bioactive peptides which may play a role as ingredients in functional foods. This study therefore sought to optimize the preparation techniques used to obtain the necessary protein hydrolysate from de-fatted lemon basil seeds (DLBS), and subsequently to examine the ACE inhibitory capabilities of the resulting hydrolysate. Response Surface Methodology (RSM) was used for the hydrolysis of DLBS by Alcalase®, with observation of the resulting ACE inhibitory activity and degree of hydrolysis (DH). The optimum conditions were 55 °C and 103 minutes with a ratio of enzyme to substrate of 7.0% w/v. The hydrolysate was fractionated by ultrafiltration and purified through RP-HPLC. The results reveal that the F2 sub-fraction demonstrated the highest ACE inhibitory activity. The amino acid sequence of this peak was identified by mass spectrometry as LGRNLPPI and GPAGPAGL with a molecular weight of 879.06 and 639.347 Dalton, respectively. These peptides were classified as non-toxic and bitter peptides. For the synthesized version of these peptides, the ACE inhibitory activity values, measured by IC50, were 0.124 ± 0.02 mM and 0.013 ± 0.001 mM, respectively. Analysis of the Lineweaver-Burk plot confirmed that these peptides served as non-competitive ones. The study of molecular docking showed that the ACE inhibitory behavior of both purified peptides was mainly due to the interactions of the hydrogen bonds between the peptides and ACE. It is therefore suggested that DLBS may be a useful raw material allowing the production of antihypertensive peptides which can offer therapeutic and commercial benefits as an ingredient in functional foods.

In this work, a defatted corn germ protein (DCGP) was pretreated with single frequency ultrasound at 20 kHz (SFU) and multi-frequency ultrasounds (20, 28, 35 and 40 kHz) (MFU). The microstructures, morphology, amino acid content, degree of hydrolysis, protein hydrolysate concentration and angiotensin-converting enzyme (ACE) inhibitory activity of the DCGP hydrolysate were analyzed. The results showed that both SFU and MFU pretreatments increased the ACE inhibitory activity of the DCGP hydrolysate more than that observed for the traditional method. Also, the SFU pretreatment showed the highest ACE inhibitory activity of 54% in comparison with that of the DCGP hydrolysates obtained via the traditional and MFU pretreatment methods. The results of ultraviolet-visible (UV-Vis) spectroscopy, fluorescence spectroscopy, surface hydrophobicity studies, Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) and scanning electron microscopy (SEM) confirmed the extent of the changes that occurred in the microstructures and morphology of DCGP pretreated with SFU and MFU. The results also indicated that the hydrophobic amino acid concentration was comparably higher in DCGP pretreated with SFU and MFU than that in the DCG protein isolated via the traditional method.

A Porphyra dioica protein extract was enzymatically hydrolysed and then fractionated using semi-preparative reverse-phase high performance chromatography. The hydrolysate and its fractions were tested for their oxygen radical absorbance capacity (ORAC) along with their angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. The most potent fraction was analysed by liquid chromatography mass spectrometry. Eight peptide sequences were selected for synthesis based on their structure-activity criteria for bioactivity. Asp-Tyr-Tyr-Lys-Arg showed the highest ORAC activity (4.27 ± 0.15 μmol Trolox equivalent per μM). Thr-Tyr-Ile-Ala had the highest ACE inhibitory activity (IC50: 89.7 ± 7.10 μM). Tyr-Leu-Val-Ala was the only peptide showing DPP-IV inhibitory activity (IC50: 439 ± 44 μM). Apart from Asp-Tyr-Tyr-Lys-Arg and Thr-Tyr-Ile-Ala, which displayed increased ORAC activity, the bioactivities of the peptides were either maintained or decreased following in vitro simulated gastrointestinal digestion. The results indicate that P. dioica-derived peptides may have potential applications as health enhancing ingredients.

Changes in the structure of tropomyosin (TM) altered the texture profiles of eel balls and the inhibitory activity of the angiotensin-converting enzyme (ACE). The secondary and tertiary structure of TM was determined after high hydrostatic pressure (HHP) treatment. The correlation between the spatial structure of TM and the texture profiles of eel balls was developed and discussed. The β-sheet was converted to a β-turn and a random coil when treated at HHP (200-400 MPa), meanwhile the α-helix unfolded and was converted into a β-sheet, β-turn and a random coil with treatment at 500 and 600 MPa. The surface hydrophobicity (H0) was increased and the sulfhydryl (SH) content decreased with an increase in the pressure. The results indicated that the texture profiles of eel balls showed a negative relationship with the α-helix, β-sheet and SH content. The texture profiles of eel balls were greatly enhanced after treatment at 500 and 600 MPa, leading to the improved surface network of the eel ball products. The ACE inhibitory activity of TM after HHP treatment exhibited a positive relationship with the β-sheet content in the protein. The ACE inhibitory activity was preserved under 600 MPa.

Hypertension is known as an important factor in cardiovascular disease. An angiotensin-I-converting enzyme (ACE) inhibitor is one of the synthetic drugs that is widely used to control hypertension. Here, we have prepared and investigated the ACE inhibitory peptide from Ruditapes philippinarum fermented with Bacillus natto. The fermented product obtained under optimized fermentation conditions exhibited ACE inhibitory activity. The ACE inhibitory peptides were purified by ultrafiltration, gel filtration chromatography and RP-HPLC, sequentially. A novel peptide with high ACE inhibitory activity (IC50 of 8.16 μM) was isolated and the amino acid sequence was identified to be Val-Ile-Ser-Asp-Glu-Asp-Gly-Val-Thr-His (VISDEDGVTH) by LC-MS/MS. The Lineweaver-Burk plots suggested that VISDEDGVTH acts as a competitive inhibitor against ACE. Our in vitro study indicated the stability of VISDEDGVTH against gastrointestinal proteases. An in vivo study in rats proved the significant anti-hypertension effect of the peptide. Furthermore, the cellular mechanism of VISDEDGVTH moderating hypertension was investigated. We found that VISDEDGVTH enhanced the release of NO, inhibited the secretion of EF-1, and scavenged ROS in human vascular endothelial cells. Our study suggests that VISDEDGVTH serves as an anti-hypertension and anti-inflammatory drug and as a beneficial ingredient in functional foods and pharmaceuticals.

Tilapia skin gelatin was hydrolyzed by successive simulated gastrointestinal digestion, and the hydrolysates were further separated by transport across a Caco-2 cell monolayer. Angiotensin-I-converting enzyme inhibitory (ACEI) peptides were separated by successive chromatographic steps from the transport hydrolysates. We have identified two key ACEI peptides, namely VGLPNSR (741.4133 Da) and QAGLSPVR (826.4661 Da) with IC50 values of ACEI activity of 80.90 and 68.35 μM, respectively. Lineweaver-Burk plots indicated that the inhibitory ACE kinetics of the two peptides were noncompetitive. Molecular docking simulation showed that the two peptides could interact with the ACE site via hydrogen bonds with high binding power. However, the hydrogen bonds were not formed with the key amino acid residues in the active site of ACE. This finding was in accordance with the noncompetitive inhibition. This study established a novel approach to identify key ACEI peptides and suggested the use of tilapia peptides as functional food ingredients to prevent hypertension.

Milk contains various bioactive components with osteoanabolic properties. This study investigates the comparative effect of the whey-derived antioxidative (YVEEL) and angiotensin-converting enzyme inhibitory (YLLF) bioactive peptides on bone remodelling in ovariectomised (OVX) osteoporotic rat model. OVX animals were administered with antioxidative (AO) (500 μg kg-1 day-1) and angiotensin-converting enzyme inhibitory (ACE inhibitory) (50 μg kg-1 day-1) peptides for eight weeks. Trabecular microarchitectural parameters of femoral and tibiae bone were determined using micro-CT scan. Bone formation, resorption, turnover markers (ALP, RANKL, OCN) and inflammatory cytokines (TNF-α, TGF-β, IFN-γ) were determined by ELISA. Both AO and ACE inhibitory peptides inhibited the increase in bone turnover and inflammatory cytokines while increased the bone formation markers. The altered morphometric parameters of femoral and tibiae bones due to OVX were strikingly attenuated by the peptide administration. The results indicated that AO peptide exerts more osteoprotective potential than ACE inhibitory peptide by suppressing inflammatory status and enhancing bone formation markers.

Herein, the ability of ginger glycoproteins to inhibit the angiotensin-converting enzyme (ACE) is characterized. The activity is monitored via HPLC, and then the crude glycoproteins are enriched with lectin microarrays and magnetic microspheres. The N-linked glycans released from the enriched glycoproteins by PNGase F are identified by MALDI-TOF-MS. The results suggest that the crude ginger glycoproteins are active against ACE with an IC50 value of 0.83 ± 0.09 mg mL-1. The ginger glycoproteins are enriched by concanavalin A (Con A) and solanum tuberosum (Potato) lectin (STL), and the structures of the N-glycans released from the ginger glycoproteins include high-mannose type glycans, fucosylated-type glycans, and hybrid-type glycans, as analyzed by MALDI-TOF-MS. The results of this study are expected to provide a reference for the glycan structure of ginger glycoproteins with ACE-inhibitory activity.

Winged bean seed (WBS) is an underutilized tropical crop. The current study evaluates its potential to reduce blood pressure (BP) in spontaneously hypertensive rats and finds that it reduces BP significantly, in a dose-dependent manner. Five peptides with the sequences, RGVFPCLK, TQLDLPTQ, EPALVP, MRSVVT and DMKP, have been characterized in terms of their stability against ACE via in vitro and in silico modelling. All peptides exhibited IC 50 values between 0.019 and 6.885 mM and various inhibitory modes, including substrate, prodrug and true inhibitor modes. The toxicity status of non-Current Good Manufacturing Practice (non-CGMP) peptides is evaluated and the results show that such peptides are toxic, and thus are not suitable to be tested in animals, particularly in repeated-dose studies. In short, WBS hydrolysate demonstrated in vitro ACE inhibitory properties and in vivo blood pressure lowering efficacy in rat models, fostering its potential as a functional food ingredient. Non-CGMP grade peptides are toxic and unfit for testing in animal models.

The objective of this work was to identify a novel ACE inhibitory peptide from myosin using a number of in silico methods. Myosin was evaluated as a substrate for use in the generation of ACE inhibitory peptides using BIOPEP and ExPASy PeptideCutter. Then the ACE inhibitory activity prediction of peptides in silico was evaluated using the program peptide ranker, following the database search of known and unknown peptides using the program BIOPEP. In addition, the interaction mechanisms of the peptide and ACE were evaluated by DS. All of the tripeptides were predicted to be nontoxic. Results suggested that the tripeptide NCW exerted potent ACE inhibitory activity with an IC 50 value of 35.5 μM. Furthermore, the results suggested that the peptide NCW comes into contact with Zn 701, Tyr 523, His 383, Glu 384, Glu 411, and His 387. The potential molecular mechanism of the NCW/ACE interaction was investigated. Results confirmed that the higher inhibitory potency of NCW might be attributed to the formation of more hydrogen bonds with the ACE's active site. Therefore, the in silico method is effective to predict and identify novel ACE inhibitory peptides from protein hydrolysates.

In this study, production of bioactive peptides with angiotensin converting enzyme (ACE) inhibitory activity from sea cucumber (Stichopus japonicus) gonad using commercial protamex was optimised by response surface methodology (RSM). As a result, the optimal condition to achieve the highest ACE inhibitory activity in sea cucumber gonad hydrolysate (SCGH) was hydrolysis for 1.95 h and E/S of 0.75%. For further characterisation, three individual peptides (EIYR, LF and NAPHMR) were purified and identified. The peptide NAPHMR showed the highest ACE inhibitory activity with IC 50 of 260.22 ± 3.71 μM. NAPHMR was stable against simulated gastrointestinal digestion and revealed no significant cytotoxicity toward Caco-2 cells. Molecular docking study suggested that Arg, His and Asn residues in NAPHMR interact with the S2 pocket or Zn 2+ binding motifs of ACE via hydrogen or π-bonds, potentially contributing to ACE inhibitory effect. Sea cucumber gonad is thus a potential resource to produce ACE inhibitory peptides for preparation of functional foods.

Ageing is widely used in the meat industry to improve tenderness mainly as a result of the breakdown of muscular proteins through the action of endopeptidases during storage time. In addition, meat contains a large pool of other proteolytic enzymes, mainly exopeptidases, which cut from the outer edges of proteins and polypeptides generating a vast array of peptides and free amino acids. Some of these peptides could potentially exert bioactivities of interest for human health. In this study, ACE-inhibitory and antioxidant activities during meat ageing in chilled-storage at 4 °C for a period of time of 0, 1, 2, 3 and 4 weeks have been determined. Beef loin steak samples were analysed before and after cooking, and gastrointestinal digestion was simulated in order to study its effect on the bioactivity. Control and cooked samples showed an increase in the ACE-inhibitory activity from the third week of chilled storage whereas non-significant differences in the antioxidant activity between control and cooked samples were detected during the studied time. After gastrointestinal digestion of samples, there was a significant increment of the ACE-inhibitory and antioxidant activities in comparison with control and cooked samples at all the studied times. As a main conclusion, cooking does not significantly influence the bioactivity detected whereas the in vitro gastrointestinal digestion produces a significant increase in the ACE-inhibitory and antioxidant activities from the first week, probably due to the intense generation of small peptides as a result of the action of gastrointestinal enzymes.

Egg proteins contain a wide set of peptide sequences which have an impact on cardiovascular health. Their modes-of-action involve, among others, the inhibition of angiotensin-converting enzyme (ACE) and antioxidant and anti-inflammatory properties. In this review, we focus particularly on ACE-inhibition and discuss recent findings in: (i) production methods for egg protein-derived ACE-inhibitory peptides, (ii) in vitro functionality of these peptides, (iii) their intestinal digestion and absorption in order to reach the target tissue, (iv) the impact of ACE-inhibitory egg-derived peptides in vivo and (v) future perspectives for the implementation of egg-derived ACE-inhibitory peptides as functional foods.

Increasing evidence on goat milk and the health benefits of its derived products beyond its nutritional value show its potential as a functional food. In this study, goat milk fractions were tested for their total antioxidant capacity using different methods (ORAC, ABTS, DPPH and FRAP), as well as their angiotensin-I-converting-enzyme inhibitory and antimicrobial (against Escherichia coli and Micrococcus luteus) activities. Different whey fractions (whey, cation exchange membrane permeate P and retentate R) of two fermented skimmed goat milks (ultrafiltered goat milk fermented with the classical starter bacteria or with the classical starter plus the Lactobacillus plantarum C4 probiotic strain) were assessed. Additionally, P fractions were divided into two sub-fractions after being passed through a 3 kDa cut-off membrane: (a) the permeate with peptides of MW <3 kDa (P < 3); and (b) the retentate with peptides and proteins of MW >3 kDa (P > 3). No differences in biological activities were observed between the two fermented milks. However, the biological peptides present in the P < 3 fraction showed the highest total antioxidant capacity (for the ORAC assay) and angiotensin-I-converting-enzyme inhibitory activity. Those present in the R fraction showed the highest total antioxidant capacity against ABTS˙ + and DPPH˙ radicals. Some antimicrobial activity against E. coli was observed for the fermented milk containing the probiotic, which could be due to some peptides being released by the probiotic strain. In conclusion, small and non-basic bioactive peptides could be responsible for most of the angiotensin-I-converting-enzyme inhibitory and antioxidant activities. These findings reinforce the potential benefits of the consumption of fermented goat milk in the prevention of cardiovascular diseases associated with oxidative stress and hypertension.

Stones from olives and Prunus genus fruits are cheap and sustainable sources of proteins and could be potential sources of bioactive peptides. The main limitation to the use of these seeds is the presence of amygdalin. This work proposes to determine amygdalin in olive and Prunus seeds and in protein isolates obtained from them. Moreover, antioxidant, angiotensin I converting enzyme (ACE) inhibitor, and hypocholesterolemic properties will be evaluated in hydrolysates obtained from these seeds. Despite some seeds contained amygdalin, all protein isolates were free of this substance. Two different procedures to obtain bioactive peptides from protein isolates were examined: gastrointestinal digestion and processing with Alcalase, Flavourzyme or Thermolysin. Higher antioxidant, ACE inhibitor and hypocholesterolemic activities were observed when proteins were processed with Alcalase, Flavourzyme or Thermolysin. The highest antioxidant and ACE inhibitor capacities were observed for the Prunus genus seed hydrolysates while the highest capacity to reduce micellar cholesterol solubility was observed for the apricot and olive seed hydrolysates.

This work was focused on the study of the bioactive potential of three fish protein hydrolysates, one of them prepared from industrial sardine by-products (head and viscera) and the others from tuna by-products (head, and muscle and viscera). These protein hydrolysates exhibited moderate ability to inhibit Angiotensin Converting Enzyme or ACE (IC50 between 0.24-1.16 mg dry weight per ml) and prolyl oligopeptidase or PO (IC50 between 3.30-9.57 mg ml(-1)), those obtained from tuna by-products being the most effective. Overall, ACE- and PO-inhibiting activities were enhanced by sequential nanofiltration through 3 and 1 kDa MWCO membranes (IC50 between 0.02-0.16 mg ml(-1) (ACE) and 1.10-4.21 mg ml(-1) (PO)). The inhibitory properties of the hydrolysates were greatly improved by in vitro gastric digestion, and were barely affected by further intestinal digestion. The digested tuna hydrolysates, mainly that from heads, proved to be the best source of PO- and ACE- inhibiting molecules (IC50 = 0.16 mg ml(-1) (ACE) and 1.04 mg ml(-1) (PO)) and could be potential new ingredients in food with interest in the prevention or treatment of cardiovascular and neurological diseases.

Three novel egg white-derived peptides were demonstrated to display in vitro activities against the angiotensin converting enzyme (ACE). A further study was conducted to assess their anxiolytic-like effects upon spontaneously hypertensive rats (SHRs) after an oral administration of the peptides, which showed the same behavioral performance as that using an elevated plus maze (EPM). In the EPM experiment, the behavioral effects of peptides TNGIIR, RVPSL, and QIGLF were investigated at doses ranging from 5 to 50 mg kg(-1), with records of the number of entries by the rats into the open arms, the time the rats spent in the open arms, and the total amount of entries into the open + closed arms. The results showed that the peptides TNGIIR and RVPSL, in a range of 5-50 mg kg(-1), exerted an anxiolytic effect on the SHRs. These results suggested that the egg white derived peptides TNGIIR and RVPSL could be considered as possible functional food or food ingredients due to their in vivo anti-stress and anti-anxiety effects on the SHRs.

Hypertension is one of the most important causes of cardiovascular and renal morbidity and mortality, and it represents a serious health problem in Western countries. Over the last few decades scientific interest in food-derived antihypertensive peptides has grown as an alternative to drugs in the control of systemic blood pressure. Most of these peptides target the angiotensin I-converting enzyme (ACE) but emerging evidence points to other antihypertensive mechanisms beyond ACE inhibition. The milk protein lactoferrin (LF) is a good source of orally active antihypertensive peptides the characterization of which, including ex vivo functional assays and in vivo approaches, shows that they might act on several molecular targets. This review summarizes the mechanisms of action underlying the blood pressure-lowering effects of LF-derived peptides, focusing on their interaction with different components of the renin-angiotensin (RAS) and endothelin (ET) systems. The ability of LF-derived peptides to modify the expression of genes encoding proteins involved in the nitric oxide (NO) pathway and prostaglandin synthesis is also described.

The methanolic extract of the wild edible mushroom Cantharellus cibarius Fr. (chanterelle) was analyzed for in vitro antioxidative, cytotoxic, antihypertensive and antibacterial activities. Various primary and secondary metabolites were found. Phenols were the major antioxidant components found in the extract (49.8 mg g(-1)), followed by flavonoids, whose content was approximately 86% of the total phenol content. Antioxidant activity, measured by four different methods, was high for inhibition of lipid peroxidation (EC50 = 1.21 mg mL(-1)) and chelating ability (EC50 = 0.64 mg mL(-1)). The antioxidant activity of the C. cibarius methanol extract was achieved through chelating iron compared to hydrogen atom and/or electron transfer. The extract showed good selectivity in cytotoxicity on human cervix adenocarcinoma HeLa, breast carcinoma MDA-MB-453 and human myelogenous leukemia K562, compared to normal control human fetal lung fibroblasts MRC-5 and human lung bronchial epithelial cells BEAS-2B. The extract had inhibitory activity against angiotensin converting I enzyme (ACE) (IC50 = 0.063 mg mL(-1)). The extract revealed selective antimicrobial activity against Gram-positive bacteria with the highest potential against E. faecalis. The medicinal and health benefits, observed in wild C. cibarius mushroom, seem an additional reason for its traditional use as a popular delicacy food.

The aim was to evaluate the angiotensin-I converting enzyme (ACE) inhibitory potential and the antioxidant capacity of pure synthesized peptides (GLTSK, LSGNK, GEGSGA, MPACGSS and MTEEY) originally identified in the non-digestible fraction (NDF) of common beans (P. vulgaris L.) that had previously demonstrated antiproliferative activity against human colorectal cancer cells. The five peptides were able to inhibit ACE with half maximal inhibitory concentration (IC50) values ranging from 65.4 (GLTSK) to 191.5 μM (MPACGSS). The combination of GLTSK and MTEEY increased the ACE inhibition by 30% compared to equieffective doses of the single peptides. According to molecular docking analysis, the five peptides had lower estimated free energy values (-6.47 to -9.34 kcal mol(-1)) when they interacted with the catalytic site of ACE than that of the substrate hippuryl-histidyl-leucine (-5.41 kcal mol(-1)), thus inhibiting the enzymatic activity. According to molecular docking analysis, the five peptides interacted with four (His353, Ala354, Glu411 and Tyr523) out of 6 catalytic residues. Moreover, MPACGSS had the highest antioxidant activity according to Ferric reducing antioxidant power (FRAP) (421.58 μmol FeSO4 mg(-1)), Fe(2+) chelation (2.01 μmol Na2EDTA mg(-1)) assays, and also in DPPH (748.39 μmol Trolox per mg of dry peptide) and ABTS (561.42 μmol Trolox mg(-1)) radical scavenging assays. The results support the hypothesis that peptides present in the non-digestible fraction of common beans (Phaseolus vulgaris L.) may exert their physiological benefits independent of their antioxidant capacity, by ACE inhibition through interaction with its catalytic cavity.

The present study was conducted to explore a novel strategy to enhance angiotensin I-converting enzyme (ACE) inhibitory activities of navy bean by preparation of navy bean milk (NBM) which was then subjected to fermentation of four lactic acid bacteria (LAB) strains, namely, Lactobacillus bulgaricus, Lactobacillus helveticus MB2-1, Lactobacillus plantarum B1-6, and Lactobacillus plantarum 70810. With the exception of L. helveticus MB2-1, the other three selected strains had good growth performances in NBM with viable counts increased to log 8.30-8.39 cfu ml(-1) during 6 h of fermentation, and thus were selected for the following investigations. Protein contents of NBM significantly reduced when treated with L. bulgaricus and L. plantarum B1-6, and the electrophoresis patterns showed the preferable proteins for LAB strains to hydrolyze were α- and β-type phaseolins, whereas γ-type phaseolin was resistant to hydrolysis. RP-HPLC analysis demonstrated all fermented NBM had higher intensities of peaks with retention times between 2.5 and 3.5 min indicative of formation of small peptides. All fermented NBM showed higher ACE inhibitory activity compared to the unfermented ones, for which 2 h, 3 h, and 5 h were found to be the optimum fermentation periods for respectively L. plantarum 70810, L. plantarum B1-6 and L. bulgaricus, with IC50 values of 109 ± 5.1, 108 ± 1.1, and 101 ± 2.2 μg protein ml(-1). The subsequent in vitro gastrointestinal simulation afforded all fermented extracts reduced IC50 values and the extracts fermented by L. plantarum B1-6 exerted the lowest IC50 value of 21 ± 2.1 μg protein ml(-1). The research has broadened our knowledge bases on the effect of LAB fermentation on the degradation of navy bean proteins and the capacity to release ACE inhibitory peptides. The approach was promising to obtain probiotic products with potential to serve as functional ingredients targeting hypertension.

In the digestive tract of humans, bioactive peptides, i.e. protein fragments impacting the physiological activity of the body, may be released during the digestion of food proteins, including those of fish. The aim of the study was to establish the method of human ex vivo digestion of carp muscle tissue and evaluate the angiotensin I-converting enzyme inhibitory and antioxidant activities of hydrolysates obtained after digestion. It was found that the hydrolysates of carp muscle tissue obtained with the three-stage method of simulated ex vivo digestion showed ACE inhibitory as well as antioxidative activities. It was demonstrated that the degree of hydrolysis depended on the duration of individual stages and the degree of comminution of the examined material. Although the applied gastric juices initiated the process of hydrolysis of carp muscle tissue, the duodenal juices caused a rapid increase in the amount of hydrolysed polypeptide bonds. The antihypertensive and antioxidative activities of the hydrolysates of carp muscle tissue increased together with progressive protein degradation. However, the high degree of protein hydrolysis does not favour an increase in the activity of free radical scavenging. The presented results are an example of the first preliminary screening of the potential health-promoting biological activity of carp muscle tissue in an ex vivo study.

Recent studies have investigated anti-hypertensive peptides derived from natural food products. In this work, we focus on Phascolosoma esculenta as a resource of anti-hypertensive peptides, which is also a seafood with high nutritive value. Compared with FAO/WHO requirements, P. esculenta was confirmed to contain high contents of amino acids and minerals. To investigate the anti-hypertensive activity of P. esculenta, water-soluble and insoluble proteins were extracted and hydrolysed by pepsin and trypsin, respectively. The hydrolysates of water-soluble proteins derived by pepsin and pepsin-trypsin exhibited angiotensin-converting enzyme (ACE) inhibitory activity with IC50 values of 0.67 and 0.24 mg ml(-1), respectively, and those of water-insoluble proteins presented IC50 values of 0.4 and 0.1 mg ml(-1), respectively. Experiments on 'spontaneously hypertensive rats' (SHRs) were carried out to test the anti-hypertension activity in vivo, which confirmed that the hydrolysates played a significant role in reducing both diastolic blood pressure (DBP) and systolic blood pressure (SBP). Consistently, the in vivo anti-hypertensive activity of the hydrolysis products of pepsin and trypsin used together was also higher than that by using pepsin hydrolysis products alone. As stated in both sets of results, we believe that P. esculenta is an excellent resource of antihypertensive peptides and warrants further investigation.

Hypertension is one of the major causes of cardiovascular-related diseases, which is highly associated with angiotensin-I-converting enzyme (ACE) activity and oxidative stress. In this study, winged bean seed (WBS), a potential source of protein, was utilised for the production of bifunctional proteolysate and biopeptides with ACE inhibitory and antioxidative properties. An enzymatic approach was applied, coupled with pretreatment of shaking and centrifuging techniques to remove endogenous ACE inhibitors prior to proteolysis. ACE inhibition reached its highest activity, 78.5%, after 12 h proteolysis while antioxidative activities, determined using assays involving DPPH˙ radical scavenging activity and metal ion-chelating activity, reached peaks of 65.0% and 65.7% at 8 h and 14 h, respectively. The said bioactivities were proposed to share some common structural requirements among peptides. A two-dimensional approach was employed for characterisation of effective peptides based on hydrophobicity, using RP-HPLC, and isoelectric property, using isoelectric focusing technique. Results revealed that acidic and basic peptides with partially higher hydrophobicity provided higher ACE inhibition activity than did neutral peptides. Finally, by using Q-TOF mass spectrometry, two peptide sequences (YPNQKV and FDIRA) with ACE inhibitory and antioxidative activities were successfully matched with a database. This study indicates that the WBS proteolysate can be a potential bifunctional food ingredient as the identified biopeptides demonstrated both ACE inhibitory and antioxidative activities in vitro.

Different monofloral honeys from Castilla-La Mancha (Spain) have been studied in order to determine their main functional and biological properties. Thyme honey and chestnut honey possess the highest antioxidant capacity, which is due to their high vitamin C (in thyme honey) and total polyphenolic content (in chestnut honey). On the other hand, chestnut honey showed high antimicrobial activity against Staphylococcus aureus and Escherichia coli, whilst others had no activity against S. aureus and showed very small activity against E. coli. Moreover it was found that the antimicrobial activity measured in chestnut honey was partly due to its lysozyme content. In addition the angiotensin I-converting enzyme (ACE) inhibitory activity was measured, and the ACE inhibition is one mechanism by which antihypertensive activity is exerted in vivo. All the types of honey showed some activity but chestnut honey had the highest ACE inhibitory activity.

Angiotensin I converting enzyme (ACE) was inhibited by the culture medium of Bacillus subtilis subsp. natto, which ferments boiled soy beans to natto, a Japanese traditional food. Subtilisin NAT (nattokinase) produced by B. subtilis also inhibited ACE, and the inhibition was markedly stimulated by heat treatment of subtilisin at 120 °C for 15 min. Inhibition of ACE by subtilisin was of a mixed type: the decrease in V(max) and the increase in K(m) value. SDS-polyacrylamide gel electrophoresis showed that heat treatment of subtilisin caused inactivation with fragmentation of the enzyme protein into small peptides. The inhibitory action of subtilisin was not due to an enzymatic action of protease, but may be ascribed to the potent ACE-inhibitory peptides such as LY and FY, amino acid sequences in subtilisin. HPLC-MS analysis of heat-inactivated subtilisin confirmed that LY and FY were liberated by fragmentation of the enzyme. Inhibition of ACE by subtilisin and its degradation peptides such as LY and FY may participate in the suppression of blood pressure by ingestion of natto.