Purification and molecular docking study of angiotensin I-converting enzyme (ACE) inhibitory peptides from hydrolysates of marine sponge Stylotella aurantium

Angioteinsin I-converting enzyme (ACE) inhibitory peptide was isolated from marine sponge (Stylotella aurantium) hydrolysate prepared by various hydrolysis enzymes. The peptic hydrolysate exhibited highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight. The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. The amino acid sequences of the purified peptides were identified to be Tyr-Arg (337.2 Da), and Ile-Arg (287.2 Da). The purified peptides from marine sponge had an IC₅₀ value of 237.2 μM and 306.4 μM, respectively. The molecular docking study revealed that ACE inhibitory activity of the purified peptides was mainly attributed to the hydrogen bond interactions and Pi interaction between the dipeptides and ACE. The results suggest that marine sponge, S. aurantium would be an attractive raw material for the manufacture of anti-hypertensive nutraceutical ingredients.     

Structure and ACE-Inhibitory Activity of Peptides Derived from Hen Egg White Lysozyme

Angiotensin I-converting enzyme plays an important role in hypertension and therefore its inhibition is considered to be a useful procedure in the prevention of hypertension. Two novel ACE inhibitory peptides were purified and identified from the papain-trypsin hydrolysate of hen egg white lysozyme using reverse phase-high performance liquid chromatography. The sequences of identified peptides were NTDGSTDYGILQINSR (MW: 1,753.98?±?0.5?Da) and VFGR (MW: 459.26?±?0.5?Da), which were named F2 and F9 peptide, respectively. Analyses of the far-UV CD spectra of ACE in the absence and presence of the F2 peptide revealed ACE secondary structural changes. In the presence of the F2 peptide, a loss of helical content of ACE was observed, which can lead to decrease of the enzymatic activity. Lineweaver?CBurk plots show that the identified peptides both act as non-competitive ACE inhibitors. These findings would be helpful on the understanding of interaction between ACE and its inhibitory peptides.     

Enzyme proteolysis enhanced extraction of ACE inhibitory and antioxidant compounds (peptides and polyphenols) from Porphyra columbina residual cake

The traditional method to obtain phycocolloids from seaweeds implies successive extraction steps with cold and hot water. The residual cake derived from phycocolloids obtaining process of red seaweed Porphyra columbina is a waste containing 27 % protein and 10.7-mg gallic acid equivalents (100 g)^?1. Seaweeds contain functional proteins, and the enzymatic hydrolysis of these proteins has been shown to release bioactive peptides. The aims of this study were to extract bioactive peptides and polyphenols after enzymatic hydrolysis of the residual cake and to evaluate their ACE inhibitory and antioxidant capacities (TEAC, DPPH, and copper-chelating activity). Residual cake hydrolysate has low molecular weight peptides containing Asp, Glu, Ala, and Leu. Residual cake hydrolysate had higher protein solubility than residual cake. ACE inhibition (≈45 %) and radical scavenging activity (TEAC and DPPH inhibition) were attributed mainly to low molecular weight peptides (500 Da) and polyphenols compounds released during proteolysis. The 50 % inhibition protein concentration value (IC50) corresponded to residual cake hydrolysate was 1.01?±?0.02 and 0.91?±?0.01 g L^?1, for ABTS and DPPH, respectively. Also, residual cake hydrolysate had high copper-chelating activity (≈97.5 %). Hydrolysis could be used as a means to obtain ACE inhibitory and antioxidant compounds (peptides and polyphenols) from algae protein waste and add value to the phycocolloids extraction process.     

Purification and Characterization of Antioxidant Peptides from Oyster (Saccostrea cucullata) Hydrolysate and the Anticancer Activity of Hydrolysate on Human Colon Cancer Cell Lines

The focus of the study was to investigate antioxidant activity and characterize antioxidant peptides from oyster (Saccostrea cucullata) protein hydrolysate. The protease hydrolysate of oyster exhibited strong potential to donate hydrogen and was able to scavenge Hydrogen peroxide, Hydroxyl and DPPH radicals. Due to the high antioxidant potential, hydrolysate was purified in Sephadex G-25 gel filtration chromatography. The active peptide fraction was further purified by UPLC-MS. Totally seven antioxidant peptides were collected. Among seven peptides (SCAP 1–7), three peptides (SCAP 1, 3 and 7) had highest scavenging ability on DPPH radicals. The amino acid sequence and molecular mass of purified antioxidant peptides (SCAP1, SCAP3 and SCAP7) were determined by Q-TOF ESI mass spectroscopy and structures of the peptides were Leu-Ala-Asn-Ala-Lys (MW = 515.29 Da), Pro-Ser-Leu-Val-Gly-Arg-Pro–Pro-Val-Gly-Lys-Leu-Thr-Leu (MW = 1,432.89 Da) and Val-Lys-Val-Leu-Leu-Glu-His-Pro-Val-Leu (MW = 1,145.75 Da), respectively. The oyster hydrolysate was tested for cell cytotoxicity on Vero (kidney epithelial cells of the African Green Monkey) and HT-29 (human colon carcinoma) cell lines. It was found that the hydrolysate did not show any cytotoxic effect for Vero cell lines and exerted a significant cytotoxic effect on HT-29 cell lines. We thus conclude that the anticancer and antioxidative hydrolysate from oyster (S. cucullata) may be useful ingredients in food and nutraceutical applications.     

A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats

Marine Chlorella ellipsoidea protein was hydrolyzed using Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, α-chymotrypsin, pepsin and papain. Alcalase-proteolytic hydrolysate exhibited the highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight (below 5 kDa, 5–10 kDa and above 10 kDa). The below 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 marine C. ellipsoidea, which was composed of 4 amino acids, Val–Glu–Gly–Tyr (MW: 467.2 Da, IC₅₀ value: 128.4 μM), was isolated. Lineweaver–Burk plots suggest that the peptide purified acts as a competitive inhibitor against ACE and stable against gastrointestinal enzymes of pepsin, trypsin and α-chymotrypsin. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHRs) also revealed that oral administration of purified peptide can decrease systolic blood pressure significantly. The results suggest that marine C. ellipsoidea would be an attractive raw material for the manufacture of antihypertensive nutraceutical ingredients.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from the marine rotifer, Brachionus rotundiformis. ACE inhibitory peptides were separated from rotifer hydrolysate prepared by Alcalase, α-chymotrypsin, Neutrase, papain, and trypsin. The Alcalase hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The IC₅₀ value of Alcalase hydrolysate for ACE inhibitory activity was 0.63 mg/ml. We attempted to isolate ACE inhibitory peptides from Alcalase prepared rotifer hydrolysate using gel filtration on a Sephadex G-25 column and high performance liquid chromatography on an ODS column. The IC₅₀ value of purified ACE inhibitory peptide was 9.64 μM, and Lineweaver–Burk plots suggest that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. Amino acid sequence of the peptide was identified as Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, with a molecular weight 1538 Da. The results of this study suggest that peptides derived from rotifers may be beneficial as anti-hypertension compounds in functional foods resource.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the Styela clava flesh tissue. Nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, α-chymotrypsin and papain) were used, and their respective enzymatic hydrolysates and an aqueous extract were screened to evaluate their potential ACE inhibitory activity. Among all of the test samples, Protamex hydrolysate possessed the highest ACE inhibitory activity, and the Protamex hydrolysate of flesh tissue showed relatively higher ACE inhibitory activity compared with the Protamex hydrolysate of tunic tissue. We attempted to isolate ACE inhibitory peptide from the Protamex hydrolysate of S. clava flesh tissue using ultrafiltration, gel filtration on a Sephadex G-25 column and high performance liquid chromatography (HPLC) on an ODS column. The purified ACE inhibitory peptide exhibited an IC₅₀ value of 37.1 μM and was identified as non-competitive inhibitor of ACE. Amino acid sequence of the peptide was identified as Ala-His-Ile-Ile-Ile, with a molecular weight 565.3 Da. The results of this study suggested that the peptides derived from enzymes-assisted extracts of S. clava would be useful new antihypertension compounds in functional food resource.     

Angiotensin I converting enzyme (ACE) inhibitory peptides from salmon byproduct protein hydrolysate by Alcalase hydrolysis

Angiotensin I converting enzyme (ACE) inhibitory peptides were produced from salmon byproduct proteins via enzymatic hydrolysis using Alcalase, flavourzyme, neutrase, pepsin, protamex and trypsin. Among them, Alcalase hydrolysate showed the highest ACE inhibitory activity, thus ACE inhibitory peptides were purified using consecutive chromatography. The purified ACE inhibitory peptides were identified to be Val-Trp-Asp-Pro-Pro-Lys-Phe-Asp (P1), Phe-Glu-Asp-Tyr-Val-Pro-Leu-Ser-Cys-Phe (P2), and Phe-Asn-Val-Pro-Leu-Tyr-Glu (P4) by time of flight-mass spectrometry/mass spectrometry (TOF-MS) analysis. The IC₅₀ values against ACE activity were 9.10 μM (P1), 10.77 μM (P2) and 7.72 μM (P4). The inhibition mode of P1, P2 and P4 was analyzed using the Lineweaver–Burk plots, demonstrating P1 to be a non-competitive inhibitor, P2 and P4 having a mixed inhibition mode. Taken together, the salmon byproduct protein hydrolysate and/or its active peptides can be used in foods for its benefits against hypertension and related diseases.     

Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacific cod (Gadus macrocephalus) skin gelatin

Potent antioxidative peptides were purified from Pacific cod (Gadus macrocephalus) skin gelatin using alcalase, neutrase, papain, trypsin, pepsin, and α-chymotrypsin. Among them, the papain hydrolysate exhibited the highest antioxidant activity. Therefore, it was further purified and obtained two peptides with amino acid sequences of Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-Gly-Asn-Val (485.5 Da). The antioxidant activity of the purified peptides was performed by electron spin resonance technique. Moreover, their intracellular free radical scavenging activity using 2′,7′-dichlorofluorescin diacetate and the protective effect against oxidation-induced DNA damage were evaluated in mouse macrophages (RAW 264.7 cells). Furthermore, both peptides have shown potential angiotensin-I converting enzyme inhibitory effect. The present study demonstrated that the peptides derived from Pacific cod (G. macrocephalus) skin gelatin could be used in the food industry as functional ingredients with potent antioxidative and antihypertensive benefits.     

Identification of a potent Angiotensin-I converting enzyme inhibitory peptide from Black cumin seed hydrolysate using orthogonal bioassay-guided fractionations coupled with in silico screening

Black cumin (Nigella sativa) seed protein (BCSP) was individually hydrolyzed with pepsin, trypsin, and α-chymotrypsin. After ultrafiltration, the α-chymotrypsin hydrolysate (< 3 kDa) exhibited the highest ACE inhibitory (ACEI) activity with an IC₅₀ value of 34.4 ± 1.5 μg/mL. This hydrolysate was orthogonally fractionalized using reversed-phase high-performance liquid chromatography (RP-HPLC) and strong cation exchange (SCX) chromatography, and the most active RP-HPLC and SCX fractions (F7 and H4, respectively) were individually screened out by ACEI assay. These two fractions were analyzed with liquid chromatography-tandem mass spectrometry (LC–MS/MS) followed by automated de novo peptide sequencing, and totally 43 ACEI candidate peptides were identified. Three overlapping peptides (VTPVGVPKW, VVTPVGVPKW, and LVLTL) were simultaneously contained in both fractions, and VTPVGVPKW (VW-9) was speculated as to the most potent ACEI peptide based on the in silico analysis. Synthetic VW-9 was used to confirm the identity, and a remarkable IC₅₀ value of VW-9 (1.8 ± 0.09 μM) was determined. Preincubation and inhibition mechanism studies indicated that VW-9 was a true inhibitor as well as a non-competitive inhibitor on ACE, which was further illustrated with the molecular docking simulation. Our study revealed that the application of VW-9 to antihypertensive products is promising.     

Antihypertensive Peptides Derived from Soy Protein by Fermentation

Soy protein is widely used as a nitrogen source in infant and adult formulations, both in an intact and hydrolyzed form. Here, the objective was to screen for maximum proteolytic activity in different strains of lactobacillus and use it for fermentation of soy protein to obtain Angiotensin converting-I-enzyme (ACE I) inhibitory peptides for its use as a nutraceutical. Based on the proteolytic activity, Lactobacillus casei spp. pseudoplantarum was selected. The two ACE inhibitory peptide fractions F2 and F3 were isolated having IC₅₀ values of 17 ± 0.63 and 30 ± 0.13 μg/ml respectively. The N-terminal sequence of peptide (F2) was determined to be Leu-Ile-Val-Thr-Gln (LIVTQ). The peptide analogues of LIVTQ were synthesized to study the effect of individual residues on ACE enzyme. LIVTQ and LIVT peptides show inhibition against ACE enzyme having an IC₅₀ value of 0.087 and 0.110 μM respectively. Our results depict that glutamine (Q) and threonine (T) residues have an important role in ACE inhibition.     

Antioxidant Activity of Purified Protein Hydrolysates from Northern Whiting Fish (Sillago sihama) Muscle

In the present study, northern whiting fish (Sillago sihama) muscle was hydrolyzed with gastrointestinal enzymes (pepsin, trypsin and α-chymotrypsin) separately and the resulted protein hydrolysates were tested for antioxidant activities using DPPH radical scavenging activity and reducing power assays. The protein hydrolysate obtained from trypsin exhibited highest antioxidant activity. Further, it was fractionated by consecutive chromatography using anion exchange and gel filtration chromatography; the separated fractions were collected and evaluated for antioxidant activity. The results showed that fraction 2 exhibited high chelating activity (73.15 % at 0.5 mg/mL) and best radical scavenging activity for DPPH radical (55.16 % at 0.5 mg/mL), ABTS radical (57.98 % at 50 μg/mL), superoxide radical (39.55 % at 200 μg/mL) and hydroxyl radical (51.33 % at 100 μg/mL). In addition, the active fraction showed strong antioxidant activity in the inhibition of linoleic acid autooxidation (60 % at 0.5 mg/mL) and also it exhibited significant protective effect on DNA damage caused by hydroxyl radicals. The size of the active fraction was found to be <360.2 Da using mass spectroscopy. These results demonstrate that muscle protein hydrolysate from northern whiting fish could be a best alternative to produce natural antioxidant peptides.     

Low-molecular weight peptides isolated from seahorse (Hippocampus abdominalis) improve vasodilation via inhibition of angiotensin-converting enzyme in vivo and in vitro

According to relevant literature, the consumption of seahorses has been used to increase male functions by improving blood flow and decreasing blood pressure. Thus, it was theorized that seahorses may also be effective against hypertension. Herein, vasodilation caused via the inhibitory effect of angiotensin-converting enzymes (ACEs) in peptides was elucidated by studying the seahorses (Hippocampus abdominalis) farmed at Jeju in South Korea. Hydrolysate was prepared using the Protamex (SHP) enzyme. The ultrafiltration system was adopted to separate certain fractions from SHP according to different molecular weights (SHP-I, MW > 10 kDa; SHP-II, MW = 5−10 kDa; SHP-III, MW < 5 kDa). The fraction with the lowest molecular weight (SHP-III) and with a low IC₅₀ value (0.044 ± 0.005) for the ACE inhibitory effect was further separated by Sephadex G-10. Three ACE inhibitory peptides from SHP-III were isolated and identified using the Q-TOF mass spectrometer (Ala-Pro-Thr-Leu, Cys-Asn-Val-Pro-Leu-Ser-Pro, and Pro-Trp-Thr-Pro-Leu). Furthermore, SHP-III-induced systolic blood pressure varied with the concentrations, as observed in the spontaneously hypertensive rat (SHR). SHP and the isolated peptides were observed to show vasodilation via ACE inhibitions and resulted in lowering the blood pressure of the SHR. These results imply that peptides from seahorses can augment male functions.     

The inhibitory activity of HL-7 and HL-10 peptide from scorpion venom (Hemiscorpius lepturus) on angiotensin converting enzyme: Kinetic and docking study

The hypertension is one of the highest risk factors for stroke, myocardial infarction, vascular disease and chronic kidney disease. Angiotensin converting enzyme (ACE) has an important role in the physiological regulation of cardiovascular system. ACE inhibition is a key purpose for hypertension treatment. In this study, two peptides named HL-7 with the sequence of YLYELAR (MW: 927.07 Da) and HL-10 with the sequence of AFPYYGHHLG (MW: 1161.28 Da) were identified from scorpion venom of H. lepturus. The inhibitory activity of HL-7 and HL-10 was examined on rabbit ACE. The inhibition mechanisms were assayed by kinetic and docking studies. The IC₅₀ values for ACE inhibition of HL-7 and HL-10 were 9.37 µM and 17.22 µM, respectively. Lineweaver-Burk plots showed that two peptides inhibited rabbit ACE with competitive manner. The molecular docking conformed experimental results and showed that the two peptides interacted with N-domain and C-domain active sites. Also, docking study revealed that the two peptides can form hydrogen and hydrophobic bonds at their binding sites. Both peptides had higher affinity to N-domain. Our results showed that HL-7 exhibited more strong interactions with amino acids at active site. It seems that HL-10 peptide could occupy more space, thereby inhibiting the substrate entrance to active site.     

Antioxidant peptides isolated from the marine rotifer,Brachionus rotundiformis

Protein derived from the rotifer Brachionus rotundiformis was hydrolyzed using different proteases (Alcalase, α-chymotrypsin, Neutrase, papain, pepsin and trypsin) for production of antioxidant peptide. Antioxidant activities of hydrolysates were evaluated using DPPH radical scavenging activity. Peptic hydrolysate exhibited the highest antioxidative activity compared to other hydrolysates. To identify antioxidant peptides, peptic hydrolysate was purified using consecutive chromatographic methods, and antioxidant peptides were identified to be Leu-Leu-Gly-Pro-Gly-Leu-Thr-Asn-His-Ala (1076 Da), and Asp-Leu-Gly-Leu-Gly-Leu-Pro-Gly-Ala-His (1033 Da) by Q-TOF ESI mass spectroscopy. EC₅₀ values of purified peptides were 189.8 and 167.7 μM, respectively. Antioxidant activities of peptides purified from the rotifer protein hydrolysate were evaluated, with results showing that peptides significantly quenched free radicals.     

Purification,modification and inhibition mechanism of angiotensin I-converting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate

Angiotensin I-converting enzyme (ACE) inhibitory peptide from silkworm pupa (Bombyx mori) was purified, modified, as well as inhibition mechanism by using molecular docking analysis. Silkworm pupa protein was hydrolyzed by neutral protease and the obtained hydrolysate was subjected to various types of chromatography to acquire peptide isolate. Then the molecular mass and amino acid sequence of the peptide was determined by MALDI-TOF/TOF MS. Subsequently, thermal and digestive stability of the peptide were explored through a high temperature processing and a simulated gastrointestinal digestion. Finally, the peptide was modified to smaller peptides and investigated their potentiate activities. Results showed that the peptide from silkworm pupa was determined to be Gly-Asn-Pro-Trp-Met (603.7 Da) with IC₅₀ 21.70 μM. Stability testing showed that ACE inhibitory activities were not significantly changed at temperature from 40 to 80 °C as well as during in vitro gastrointestinal digestion. The inhibitory activity of four modified peptides were Trp-Trp > Gly-Asn-Pro-Trp-Trp > Asn-Pro-Trp-Trp > Pro-Trp-Trp, and the IC₅₀ of Trp-Trp was 10.76 μM Docking simulation revealed that the inhibitory activity was closely related to the spatial structure of peptide and zinc ions. The purified peptide and four modified peptides may be beneficial as functional food or drug for treating hypertension.     

A heptameric peptide isolated from the marine microalga <Emphasis Type="Italic">Pavlova lutheri</Emphasis> suppresses PMA-induced secretion of matrix metalloproteinase-9 through the inactivation of the JNK,p38, and NF-κB pathways in human fibrosarcoma cells

In this study we isolate a heptameric peptide from the marine microalga Pavlova lutheri and examine its inhibitory effect and mechanism of action on the matrix metalloproteinase-9 (MMP-9) protein in human fibrosarcoma HT1080 cells. Through consecutive rounds of purification we isolated a potent MMP-9 inhibitory peptide composed of seven amino acids: Leu-Leu-Ala-Pro-Pro-Glu-Arg (796.4 Da). We show that the mRNA and protein expression levels of MMP-9 are reduced in the presence of this purified peptide. Furthermore, we show that this peptide inhibits PMA-induced translocation of nuclear factor-κB (NF-κB) by suppressing IκB-α phosphorylation and that it suppresses the phosphorylation of the c-Jun N-terminal (JNK) and p38 mitogen-activated protein kinases (MAPKs). Our results indicate that this peptide, purified from P. lutheri, has the potential to be a valuable natural preventive agent against MMP-related diseases.     

Analysis of novel angiotensin-I-converting enzyme inhibitory peptides from protease-hydrolyzed marine shrimp Acetes chinensis.

Acetes chinensis is an underutilized shrimp species thriving in the Bo Hai Gulf of China. In a previous study, we had used the protease from Bacillus sp. SM98011 to digest this kind of shrimp and found that the oligopeptide-enriched hydrolysate possessed antioxidant activity and high angiotensin I-converting enzyme (ACE) inhibitory activity with an IC50 value of 0.97 mg/ml. In this paper, by ultrafiltration, gel permeation chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC), five peptides with high ACE inhibitory activity were purified from the shrimp hydrolysates and their sequences were identified by amino acid composition analysis and molecular weight (MW) analysis. Three of them, FCVLRP (a), IFVPAF (f) and KPPETV (j), were novel ACE inhibitory peptides. Their IC50 values were 12.3 microM, 3.4 microM and 24.1 microM, respectively, and their recoveries were 30 mg/100 g (solid basis of shrimp), 19 mg/100 g and 33 mg/100 g, respectively. Lineweaver-Burk plots for the three novel peptides showed that they are all competitive inhibitors. To test the ACE inhibitory activity of peptide a, f, j after they were digested by digestive enzymes in vivo, 12 derived peptides from FCVLRP and IFVPAF were synthesized based on their amino acid sequences and the cleavage sites of digestive enzymes. No digestive enzyme cleavage site was found in KPPETV. The IC50 values of the derived peptides were determined and the result showed that except for VPAF, FC and FCVL, the ACE inhibitory activity of the other nine derived peptides did not significantly change when compared with their original peptides. Surprisingly, five peptides had lower IC50 values than their original peptides, particularly for RP (IC50 value = 0.39 microM), which is about 30 times lower than its original peptide and almost the lowest IC50 value for ACE inhibitory peptides reported. Therefore, the novel peptides identified from A. chinensis hydrolysates probably still maintain a high ACE inhibitory activity even if they are digested in vivo. This is the first report about novel ACE inhibitory peptides from hydrolysates of marine shrimp A. chinensis. The novel peptides from hydrolysate of A. chinensis and some of their derived peptides with high ACE inhibitory activity probably have potential in the treatment of hypertension or in clinical nutrition.     

Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks (<Emphasis Type="Italic">Lassi</Emphasis>) Fermented by <Emphasis Type="Italic">Lactobacillus acidophillus</Emphasis> with Consideration of Incubation Period and Simmering Treatment

The lassi, fermented milks product containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus acidophilus NCDC-15 and the incubation period and simmering effect was also optimized for production of ACE-inhibitory peptides. The time–temperature combination for the heat treatment was optimized using RSM. The biological activity was measured in the supernatant of the fermented milk after centrifugation. The lowest IC₅₀ values for the inhibition of angiotensin-converting enzyme (ACE) was found 28.9 ± 0.95 μg protein/ml in the supernatant of milk fermented by L. acidophilus and heated at 78 °C for 10 h. The fractions which showed the highest ACE-inhibitory indexes were further purified by different techniques including solid phase extraction, RP-HPLC and FPLC and the related peptides were identified by LC–MS/MS using the Ultimate 3000 nano HPLC system (Dionex) coupled to a 4000 Q TRAP electro-spray ionization mass spectrometry. The high ACE-inhibitory activity containing fractions of the milk fermented by L. acidophilus contained the sequences of b-casein (b-CN) fragment. The fraction-III showed minimum IC₅₀ value i.e. 14.57 ± 0.72 μg/ml compared with fraction-I and fraction-II. Among these peptides 14 peptides have been identified from the fraction-I of the lassi prepared from L. acidophilus i.e. β-CN f47–56, β-CN f47–57, β-CN f199–209, β-CN f176–182, β-CN f176–183, β-CN f176–184, β-CN f1–7, β-CN f57–68, β-CN f166–175, β-CN f195–206, β-CN f195–207, β-CN f195–209, β-CN f94–106 and β-CN f169–176 showed partially or completely homology to that the milk protein bioactive peptides having ACE inhibitory. The two peptides KVLPVPQK (β-CN f169–176) and YQEPVLGPVRGPFPIIV (β-CN f193–209) have the same sequence as ACE inhibitory peptides (Maeno et al. in J Dairy Sci 79(8):1316–1321, 1996; Yamamoto et al. in J Dairy Sci 77:917–922, 1994b).     

Identification and Inhibitory Mechanism of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Bovine Hemoglobin

Angiotensin I-converting enzyme (ACE, EC.3.4.15.1) inhibitory peptide is an efficacious therapy for hypertension. In this study, four dipeptides, TY, FD, FL and FG, were identified from the desalted fraction of bovine hemoglobin hydrolysate, obtained by in vitro simulated gastrointestinal digestion, via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The IC₅₀ value of TY and FL are 96.43?±?6.17 and 290.66?±?57.92 μM, respectively. The result of molecular docking indicated that TY occupied the ACE subsite S1 and S1′ with a lowest estimated binding energy of ?9.96 Kcal/mol, while FL occupied the subsite S5 with a lowest estimated binding energy of ?9.37 Kcal/mol. The subsite S1′ and S2′ are closer to the ACE active center (Zn²⁺) than S5, and the lowest estimated binding energy of TY is lower than that of FL. This work provided new ACE-inhibitory peptides derived from bovine hemoglobin hydrolysate and explained their inhibitory mechanism.