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Article pubs.acs.org/JAFC

Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Peptides with Potential Cardioprotective Effects Derived from DryCured Ham Byproducts Marta Gallego,† Leticia Mora,*,† Maria Hayes,‡ Milagro Reig,§ and Fidel Toldra†́ †

Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Avenue Agustín Escardino 7, 46980 Paterna (Valencia), Spain Teagasc, The Irish Agricultural and Food Development Authority, Food BioSciences Department, Ashtown, Dublin 15, Ireland § Instituto de Ingeniería de Alimentos para el Desarrollo, Universitat Politècnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain Downloaded via UNIV OF NEW ENGLAND on January 16, 2019 at 15:22:31 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

ABSTRACT: The interest in using food byproducts as a source of bioactive peptides has increased significantly in the recent years. The goal of this work was to determine the presence and stability of peptides showing angiotensin I-converting enzyme (ACE-I), endothelin-converting enzyme (ECE), dipeptidyl peptidase-IV (DPP-IV), and platelet-activating factoracetylhydrolase (PAF-AH) inhibitory activity derived from dry-cured ham bones, which could exert cardiovascular health benefits. ACE-I and DPP-IV inhibitory peptides were stable against heating typically used in Mediterranean household cooking methods and also to in vitro digestion. PAF-AH inhibitory activity significantly increased following simulated gastrointestinal digestion whereas ECE inhibitory significantly decreased (P < 0.05). The mass spectrometry analysis revealed a notable degradation of hemoglobin-derived peptides after simulated digestion, and the release of a large number of dipeptides that may have contributed to the observed bioactivities. These results suggest that natural peptides from Spanish dry-cured ham bones could contribute to a positive impact on cardiovascular health. KEYWORDS: ham bones, bioactive peptides, cooking, in vitro digestion, mass spectrometry

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INTRODUCTION Hypertension, diabetes, obesity, alterations in lipid metabolism, and thrombotic disorders are risk factors in the development of cardiovascular diseases, which are the most important causes of death in the world. Some of these factors can be controlled by regulating enzymatic pathways through the ingestion of food-protein-derived hydrolysates and peptides with different types of bioactivity.1,2 In fact, the main activities that would permit the complete characterization of the peptides generated in these hydrolysates are angiotensin I-converting enzyme (ACE-I), endothelin-converting enzyme (ECE), dipeptidyl peptidase-IV (DPP-IV), and platelet-activating factor-acetylhydrolase (PAF-AH) inhibitory activity. Bioactive peptides contain few amino acids rests that have a positive health impact on the human body, including antihypertensive, antioxidant, antidiabetic, and antithrombotic activities, among others.3 These peptides are inactive in the parent protein but can be released after proteolytic digestion and exert physiological effects. For this reason, they should be able to resist degradation by gastrointestinal proteases and cross the intestinal barrier and are active when they reach their target sites.4 Bioactive peptides can be generated from parent proteins using chemical or enzymatic hydrolysis, microbial fermentation, or gastrointestinal digestion but also during food processing conditions such as cooking, freezing, storage, pH variations, and physical treatments.2,5 Meat processing generates huge amounts of byproducts including bones, meat trimmings, blood, skins, and horns, which represent high economic and environmental costs.6 Some of these byproducts constitute part of the human diet since the consumption of internal organs with high nutritive © XXXX American Chemical Society

value is a normal practice in certain countries. Additionally, blood is used in traditional Asian and European foods for its technological properties and rinds and bones in Mediterranean cooking to add flavor to soups, stews, and broths.6,7 Several recent studies have reported the presence of bioactive peptides generated by enzymatic hydrolysis, processing conditions, and gastrointestinal digestion of meat byproducts. Peptides identified to date have reported antihypertensive, antioxidant, antimicrobial, opioid, and other bioactivities.2,8−10 Bones are an important source of collagen protein, which has been described to contain in its sequence bioactive peptides with potential health benefits, especially antioxidant and ACE inhibitory activities.11 Blood is also a rich protein byproduct which has been reported to show antioxidant, antihypertensive, antimicrobial, and opioid activities in hemoglobin and plasma hydrolysates.12 The purpose of this work was to determine the presence of bioactive peptides with potential cardiovascular health benefits derived from dry-cured ham byproducts as well as their stability under different acid conditions and heating simulating traditional cooking in Mediterranean households. Furthermore, in vitro-simulated digestion was used to mimic what happens to these peptides in the human gut and to assess if gastrointestinal digestion can assist in the generation of bioactive peptides. Received: October 26, 2018 Revised: December 13, 2018 Accepted: December 15, 2018

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DOI: 10.1021/acs.jafc.8b05888 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

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mixed with 200 μL of enzyme (0.125 μg/mL ECE-I in 100 mM MES with 150 mM NaCl, pH 6) and 125 μL of substrate (20 mM FPS V in 100 mM MES with 150 mM NaCl, pH 6). The fluorescence was measured after 20 min of incubation at 355 nm excitation and 405 nm emission wavelengths and results given as percent. The assay was done in triplicate, and the positive control was phosphoramidon (10 μM). The IC50 values were determined by regression analysis of inhibitory activity (%) versus sample concentration in triplicate. DPP-IV Inhibitory Activity. The assay was carried out as described by Gallego et al.15 This assay measures the fluorescence generated by 7-amido-4-methylcoumarin (AMC) when released from the fluorescent substrate Gly-Pro-AMC by the enzyme. A 50 μL amount of each sample (5 mg/mL) was added with 50 μL of enzyme (4.55 mU/mL DPP-IV in 50 mM Tris HCl buffer, pH 8.0). The reaction was initiated by adding 200 μL of substrate (0.25 mM GlyPro-AMC in 50 mM Tris HCl buffer pH 8.0, 0.5 mM DTT, 1 mM EDTA) and then incubated for 20 min at 37 °C, and the fluorescence was measured using 355 and 460 nm as excitation and emission wavelengths, respectively. Samples were assayed in triplicate, and the positive control was diprotin A (100 μM). Results were expressed as DPP-IV inhibition percentage. PAF-AH Inhibitory Activity. The assay is based on the capacity of the enzyme PAF-AH to hydrolyze the substrate 2-thio PAF, which generates free thiols that can be colorimetrically detected using 5,5′dithio-bis(2-nitrobenzoic acid) (DTNB). For that purpose, samples were assayed at 5 mg/mL using a PAF-AH inhibitor assay kit. The assay was done in triplicate and the results given as % PAF-AH inhibition achieved by the peptides. Identification of Peptides by Q/ToF Mass Spectrometry. Samples of H2O (control) and samples cooked at 100 °C for 1 h, before and after the in vitro digestion, were prepared at 0.3 mg/mL in 0.1% trifluoroacetic acid (TFA) and analyzed by nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS). A 5 μL amount of each sample was injected into a Nano-LC Ultra 1D Plus system (Eksigent AB Sciex, Dublin, CA), where it was preconcentrated onto a 75 μm × 15 cm i.d., 3 μm, C18-CL trap column (Eksigent AB Sciex) at a 3 μL/min flow rate for 5 min and using 0.1% TFA in water as mobile phase. The sample was then loaded into a 72 μm × 12 cm i.d., 3 μm, C18-CL capillary column (Nikkyo Technos Co. Ltd., Tokyo, Japan) using 0.1% formic acid (FA) in water as solvent A and 0.1% FA in acetonitrile as solvent B. A linear gradient from 5% to 35% of solvent B in 30 min was run at a 0.30 μL/min flow rate and 30 °C for peptide elution. Then peptides were analyzed in a quadrupole/time-of-flight (Q/ToF) TripleTOF 5600+ system (AB Sciex Instruments, Framingham, MA) with a nanoelectrospray ionization (nESI) system. The Q/TOF operated in positive polarity and DDA mode. Survey MS1 scans were acquired from m/z 150 to 1250 for 250 ms, and MS2 experiments were from m/z 100 to 1250 for 50 ms on the 25 most intense double- to quintuple-charged ions. Data analysis XLSTAT 2011 v5.01 software (Addinsoft, Barcelona, Spain) was used for statistical analysis of enzymatic inhibition.The statistical analyses done were one-way analysis of variance (ANOVA) and Fisher’s multiple range tests. ANOVA was performed for DPP-IV and PAF-AH inhibitory activities in order to elucidate the differences among all samples (including the different treatments before and after digestion). Results were expressed as the mean of 3 replicates ± standard deviations, and differences were considered significant at P < 0.05. Data were processed using ProteinPilot v4.5 search engine (AB Sciex, Framingham, MA) for the identification of peptides and their protein of origin. The Paragon algorithm of ProteinPilot was used to search in Uniprot database, selecting mammalian taxonomy and no enzyme specificity. The search of sequences of bioactive peptides previously identified was done using the BIOPEP database.11

MATERIALS AND METHODS

Chemicals and Reagents. Enzymes used in the simulated gastrointestinal digestion were obtained from Sigma-Aldrich, Co. (St. Louis, MO). With regard to chemicals used for enzymatic inhibition assays, angiotensin I-converting enzyme (ACE-I) from rabbit lung, phosphoramidon disodium salt, 4-morpholineethanesulfonic acid (MES), captopril, dipeptidyl peptidase-IV (DPP-IV) from porcine kidney, diprotin A, Gly-Pro-7-amido-4-methylcoumarin hydrobromide (Gly-Pro-AMC), and DL-dithiothreitol (DTT), were from Sigma-Aldrich, Co. Ethylenediaminetetraacetic acid disodium salt 2́ hydrate (EDTA) was from Panreac Quimica, S.L.U. (Barcelona, Spain), and o-aminobenzoylglycyl-p-nitro-L-phenylalanyl-L-proline (Abz-Gly-p-nitro-Phe-Pro-OH) trifluoroacetate salt was from Bachem AG (Bubendorf, Switzerland). Recombinant human endothelin-I converting enzyme (ECE-I) and fluorogenic peptide substrate V (FPS V) were from R&D Systems (Minneapolis, MN). The plateletactivating factor-acetylhydrolase (PAF-AH) inhibitory assay kit was purchased by Cayman Chemical Co. (Ann Arbor, MI). All other products were analytical grade. Preparation of Samples and Extraction of Peptides. Bones of Spanish dry-cured ham were obtained from white-breed pigs. Drycured ham bones (50 g) were minced and processed according to the method of Gallego et al.10 in triplicate. Briefly, one sample was homogenized in 200 mL of H2O as control sample; one sample was mixed with 200 mL of 0.5 N HCl, and another sample was mixed with 0.01 N HCl; two samples were prepared simulating Mediterranean household cooking: samples in 200 mL of water heated at 100 °C for 20 min and 100 °C for 1 h, respectively. After the extraction of peptides (8 min, 4 °C, overnight) using a stomacher (IUL, Barcelona, Spain), samples were centrifuged (12 000g, 20 min, 4 °C), deproteinized (3 volumes ethanol, 20 h, 4 °C), and centrifuged again (12 000g, 10 min, 4 °C). Supernatants were dried using a rotatory evaporator and lyophilized. All samples were prepared in triplicate. Simulated Gastrointestinal Digestion in Vitro. Samples (200 mg/mL) were digested following the procedure described by Gallego et al.10 Thus, salivary α-amylase (27 U/mL) was used to simulate the oral phase (3 min, 37 °C, pH 7), porcine pepsin (2000 U/mL) for the gastric phase (2 h, 37 °C, pH 3), and a mixture of trypsin (100 U/mL), chymotrypsin (25 U/mL), porcine pancreatic α-amylase (200 U/mL), porcine pancreatic lipase (2000 U/mL), and porcine bile extract (10 mM) for the intestinal phase (2 h, 37 °C, pH 7). The process was stopped by heating 2 min at 95 °C, and then it was deproteinized with ethanol (3 volumes at 4 °C and during 20 h) and centrifuged (12 000g, 10 min, 4 °C). Finally, the ethanol of the supernatant was evaporated and sample lyophilized. Analysis was done in triplicate. ACE-I Inhibitory Activity. The ACE-I inhibitory activity of the samples was determined according to the methodology of Sentandreu and Toldrá,13 which determines the capacity of ACE-I enzyme to hydrolyze the fluorescent substrate Abz-Gly-Phe(NO2)Pro. For that 50 μL of sample at different concentrations (0.016, 0.16, 0.36, 0.73, 1.46, 2.5, 4.16, and 8.3 mg/mL) and 50 μL of enzyme (3 mU/mL ACE-I in 150 mM Tris base buffer, pH 8.3) were mixed. The reaction started by adding 200 μL of substrate (0.45 mM Abz-Gly-p-nitro-Phe-Pro-OH in 150 mM Tris base buffer with 1.125 mM NaCl, pH 8.3). After 45 min of incubation at 37 °C, the fluorescence was measured at 355 nm excitation and 405 nm emission wavelengths and results given as percent. The assay was done in triplicate, and the positive control was captopril (10 μM). The IC50 values for each sample were determined by regression analysis of inhibitory activity (%) versus sample concentration in triplicate. ECE Inhibitory Activity. Inhibition of ECE activity was assessed using synthetic FPS V and quantitating the 7-methoxycoumarin reaction product group with fluorescence detection. The assay was done following the procedure described previously by FernándezMusoles et al.14 with slight changes. Thus, 25 μL of each sample at different concentrations (0.01, 0.1, 0.5, 1, and 1.5 mg/mL) was

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RESULTS AND DISCUSSION Samples from dry-cured ham bones were subjected to different treatments simulating traditional Mediterranean B


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Figure 1. ACE-I inhibitory activity of the samples H2O (control), 0.5 N HCl, 0.01 N HCl, 100 °C 20 min, and 100 °C 1 h (A) before and (B) after in vitro gastrointestinal digestion. Values represent means of three replicates, and bars represent standard deviations. Table shows the calculated IC50 value for each sample.

the other hand, two ACE-I inhibitory peptides from bovine Achilles tendon collagen were reported to retain most of their activity after the action of digestive enzymes.21 ECE Inhibitory Peptides. The endothelin system participates in the regulation of blood pressure as ECE releases the peptide endothelin (ET)-1 that binds with different receptors and induces vasoconstriction among other physiological effects. Thus, ECE inhibitors and ET receptor agonists are currently being used in treatments against hypertension, and recent studies are dealing with the evaluation of food peptides on the endothelin system.22,23 This work showed some peptides from ham bones with inhibitory effect on ECE activity (Figure 2). Samples cooked at 100 °C for 20 min and 1 h showed the best ECE inhibition before digestion, noting the low IC50 value (38 μg/mL) obtained for the sample cooked for 20 min. Samples extracted under acidic conditions did not present this bioactivity. After in vitro digestion (Figure 2B), there was a 50% reduction in the ECE inhibitory activity of the control and cooked samples while the samples treated with HCl showed 20% inhibition of ECE. These results would suggest that the breakdown of the ECE inhibitory peptides in the control and cooked samples occurred as well as a possible generation of novel peptides in the acidic samples due to the enzymatic action during gastrointestinal digestion. DPP-IV Inhibitory Peptides. DPP-IV enzyme degrades glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP), which are incretin hormones that potentiate the release of glucose-stimulated insulin, suppress the

cooking and in vitro digestion to determine the presence of bioactive peptides as inhibitors of the enzymes ACE-I, ECE, DPP-IV, and PAF-AH. These enzymes are involved in metabolic pathways that regulate the cardiovascular system, so their inhibition can result in the reduction of high blood pressure and alleviation of disorders including type 2 diabetes, obesity, atherosclerosis, and inflammatory diseases.16,17 ACE-I Inhibitory Peptides. The ACE-I enzyme is involved in the renin-angiotensin-aldosterone system (RAAS) by cleaving angiotensin-I into the vasoconstrictor angiotensin-II and induces aldosterone release. ACE-I also inactivates the vasodilatory protein bradykinin. Thus, ACE-I inhibitory peptides are widely used for treating hypertension and inflammation-associated cardiovascular diseases.18 Peptide extracts from bones were analyzed for ACE-I inhibitory activity, showing few differences after the treatments (Figure 1). The sample cooked at 100 °C for 1 h showed ACE-I IC50 inhibition values of 279 and 592 μg/mL before and after digestion, respectively. Sample treated with 0.5 N HCl prior to digestion showed approximately 30% less ACE-I inhibitory activity than the rest of the samples, which presented similar inhibition percentages but slightly lower IC50 values after digestion. These results show the stability of ACE-I inhibitory activity during cooking and following gastrointestinal digestion. To date, some studies have identified ACE-I inhibitory peptides released from the hydrolysis of meat byproducts such as chicken skin or porcine liver, showing blood pressurelowering effects in spontaneously hypertensive rats.19,20 On C


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Figure 2. ECE inhibitory activity of the samples H2O (control), 0.5 N HCl, 0.01 N HCl, 100 °C 20 min, and 100 °C 1 h (A) before and (B) after in vitro gastrointestinal digestion. Values represent means of three replicates, and bars represent standard deviations. Table shows the calculated IC50 values for each sample.

Figure 3. DPP-IV inhibitory activity of the samples H2O (control), 0.5 N HCl, 0.01 N HCl, 100 °C 20 min, and 100 °C 1 h before and after in vitro gastrointestinal digestion. Values represent means of three replicates, and bars represent standard deviations. Bar letters indicate significant differences among the values at P < 0.05.

of the acidic samples after enzymatic action increased to a lesser degree. No significant differences were found between cooked samples before and after digestion, but the DPP-IV activity showed an increase compared to the control sample prior to digestion. Cooking of dry-cured ham bones and digestion may lead to the formation of novel DPP-IV inhibitory peptides, which agrees with previous in silico

secretion of glucagon, and improve plasma glucose concentrations. As a result, DPP-IV inhibitors are good candidates for type 2 diabetes and hypertension new therapies.24,25 The DPP-IV inhibitory activity of peptides from dry-cured ham bones was lower than 30% in all samples (Figure 3). There was a notable increase (20%) in the inhibition value in the control sample after in vitro digestion, whereas the activity D


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Figure 4. PAF-AH inhibitory activity of the samples H2O (control), 0.5 N HCl, 0.01 N HCl, 100 °C 20 min, and 100 °C 1 h before and after in vitro gastrointestinal digestion. Values represent means of three replicates, and bars represent standard deviations. Bar letters indicate significant differences among the values at P < 0.05.

Figure 5. Distribution of the peptides identified by nLC-MS/MS in the samples: (A) H2O (control) before digestion, (B) 100 °C 1 h before digestion, (C) H2O (control) after digestion, and (D) 100 °C 1 h after digestion, according to their protein of origin.

analysis that showed the potential of meat byproducts proteins such as serum albumin, hemoglobin, and collagen as sources of these bioactive peptides.26 PAF-AH Inhibitory Peptides. The PAF-AH enzyme catalyzes the pro-inflammatory phospholipid mediator PAF,

which participates in several inflammatory and vascular diseases. Recent works have focused on the identification of food-derived peptides showing PAF-AH inhibition as they are promising therapeutic targets for the prevention of atherosclerotic lesions.27,28 E


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Table 1. Peptides from Hemoglobin Protein Identified by nLC-MS/MS in Samples H2O and 100 °C 1 h before and after in Vitro Digestion before digestionc protein subunit

peptide number

Alpha

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

peptide sequence AAHHPDDF AAHHPDDFN AAHHPDDFNPS AAHHPDDFNPSV AAHHPDDFNPSVH AAHHPDDFNPSVHA AAHHPDDFNPSVHAS AAHHPDDFNPSVHASL AAHHPDDFNPSVHASLD AAHHPDDFNPSVHASLDK AAHHPDDFNPSVHASLDKF AGAHGAEALER AGAHGAEALERM AHELRVDPV AHGAEALER AHHPDDFN AHHPDDFNPS AHHPDDFNPSV AHHPDDFNPSVH AHHPDDFNPSVHA AHHPDDFNPSVHAS AHHPDDFNPSVHASLD AHHPDDFNPSVHASLDK AHHPDDFNPSVHASLDKF AVGHLDDLPGALS AWGKVGGQAGAHGA DDFNPSVH DDFNPSVHA DDFNPSVHAS DDFNPSVHASLD DDFNPSVHASLDK DDLPGALSALSDL DFNPSVH DFNPSVHASLDK DPVNFK FLGFPTTKT GAHGAEALER GAHGAEALERM GGQAGAHGAEAL GGQAGAHGAEALE GGQAGAHGAEALER GGQAGAHGAEALERM GHLDDLPGALS GKVGGQAGAHGAEA GKVGGQAGAHGAEAL GKVGGQAGAHGAEALE GKVGGQAGAHGAEALER GKVGGQAGAHGAEALERM GQAGAHGAEALE GQAGAHGAEALER GQAGAHGAEALERM HGAEALER HGAEALERM HGQKVADALTKA HHPDDFNP HHPDDFNPS HHPDDFNPSV

modification

oxidation (W)

oxidation (M)

oxidation (M)

oxidation (M)

oxidation (M) oxidation (M)

F

obsd (m/z)a

calcd MW (Da)b

charge (+)

455.194 512.216 604.246 436.198 481.846 505.530 534.547 572.249 610.601 490.197 526.972 541.245 404.812 518.267 477.202 476.658 568.719 618.264 458.163 481.846 510.862 586.916 472.436 509.209 632.822 428.166 465.658 501.185 544.708 658.788 722.854 643.823 408.135 443.840 360.131 506.243 505.717 386.465 519.720 584.253 441.839 490.860 547.750 605.289 661.843 484.540 536.586 585.608 555.742 633.806 471.848 441.679 515.212 413.517 489.671 533.194 582.738

908.378 1022.421 1206.505 1305.574 1442.633 1513.670 1600.702 1713.786 1828.813 1956.908 2103.976 1080.531 1211.572 1034.551 952.473 951.383 1135.468 1234.537 1371.596 1442.633 1529.665 1757.776 1885.871 2032.939 1263.646 1281.621 929.388 1000.425 1087.457 1315.568 1443.663 1285.640 814.361 1328.636 718.365 1010.544 1009.494 1156.530 1037.489 1166.532 1322.633 1469.668 1093.540 1208.590 1321.674 1450.716 1606.818 1753.853 1109.510 1265.611 1412.647 881.436 1028.471 1237.678 977.399 1064.431 1163.500

2 2 2 3 3 3 3 3 3 4 4 2 3 2 2 2 2 2 3 3 3 3 4 4 2 3 2 2 2 2 2 2 2 3 2 2 2 3 2 2 3 3 2 2 2 3 3 3 2 2 3 2 2 3 2 2 2

H2O

100 °C, 1h

after digestiond H2O × ×

100 °C, 1h ×

× × × × × ×

× × × × × × × × × × × × × ×

× × × × × × × × × × × × × × × × ×

× × × × × × × × × × × × × × × × × × × × ×

× ×

× × × × × × × ×

×

× × × × × × × × ×

× × ×

× × ×

× × × ×

×

×

× × × × × × × ×

× ×

× ×

×


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Journal of Agricultural and Food Chemistry Table 1. continued before digestionc protein subunit

peptide number

peptide sequence

58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

HHPDDFNPSVH HHPDDFNPSVHA HHPDDFNPSVHAS HHPDDFNPSVHASLD HHPDDFNPSVHASLDK HLDDLPGA HLDDLPGAL HLDDLPGALS HLDDLPGALSA HPDDFNPS HPDDFNPSVH HPDDFNPSVHA HPDDFNPSVHAS HPDDFNPSVHASLDK KLRVDPV KVGGQAGAHGA KVGGQAGAHGAEA KVGGQAGAHGAEALE KVGGQAGAHGAEALER KVGGQAGAHGAEALERM LAAHHPDDFNPSVH LANVSTVL NLSHGSDQV

81 82

NLSHGSDQVK NLSHGSDQVKA

83 84 85 86 87 88

NLSHGSDQVKAHGQK NLSHGSDQVKAHGQKV NLSHGSDQVKAHGQKVA NLSHGSDQVKAHGQKVAD NLSHGSDQVKAHGQKVADA NVSTVLTSKY

89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113

PDDFNPSVH PTTKTYFPH QAGAHGAEALER QYGAEALER QYGAEALERM SAADKANVK SAADKANVKAA SHGSDQVKAHGQK SHGSDQVKAHGQKV SHGSDQVKAHGQKVAD TKAVGHLDDLPG VADALTKA VGGQAGAHGAEA VGGQAGAHGAEAL VGGQAGAHGAEALE VGGQAGAHGAEALER VGGQAGAHGAEALERM VGHLDDLPG VGHLDDLPGA VGHLDDLPGAL VGHLDDLPGALS VGHLDDLPGALSA VLSAADKA VLSAADKANV VLSAADKANVK

modification

deamidation (N) deamidation (N)

deamidation (N)

oxidation (M)

carbamylation

G

100 °C, 1h

H2O

100 °C, 1h

× × × × × × × ×

×

×

×

× ×

× ×

obsd (m/z)a

calcd MW (Da)b

charge (+)

651.282 458.163 487.180 563.232 605.937 419.158 475.710 519.234 554.762 464.652 582.738 618.275 661.792 560.243 413.709 489.715 589.777 710.871 517.574 575.258 519.578 408.690 479.178

1300.558 1371.596 1458.628 1686.739 1814.834 836.403 949.487 1036.519 1107.556 927.372 1163.500 1234.537 1321.569 1677.775 825.507 977.504 1177.584 1419.711 1549.796 1722.847 1555.717 815.475 956.420

2 3 3 3 3 2 2 2 2 2 2 2 2 3 2 2 2 2 3 3 3 2 2

× × × × ×

362.124 386.137

1083.531 1155.552

3 3

× ×

×

535.913 568.943 592.624 630.977 491.213 556.768

1604.802 1703.870 1774.907 1889.934 1960.972 1111.576

3 3 3 3 4 2

× × × ×

× × × × ×

514.194 364.463 596.774 518.741 592.276 452.203 523.255 460.190 493.219 555.255 408.166 394.675 512.712 569.264 633.798 474.867 518.557 461.693 497.253 553.794 597.296 632.822 387.666 515.746 558.300

1026.441 1090.545 1191.563 1035.499 1182.534 902.482 1044.556 1377.675 1476.743 1662.807 1221.635 787.444 1023.473 1136.557 1265.600 1421.701 1552.742 921.456 992.493 1105.577 1192.609 1263.646 773.428 1029.546 1114.635

2 3 2 2 2 2 2 3 3 3 3 2 2 2 2 3 3 2 2 2 2 2 2 2 2

H2O

× × × × × × ×

×

×

after digestiond

× × × × × × ×

×

×

×

× ×

× ×

× ×

×

× × × ×

× × × × × × × × × × × × ×

× × × × × × × × × × × × × × × ×

× × × × × × ×


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beta

peptide number

peptide sequence

114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171

VLSAADKANVKA VLSAADKANVKAA VLSAADKANVKAAWGK VLSAADKANVKAAWGKVGGQAGA WGKVGGQAGA WGKVGGQAGAHG WGKVGGQAGAHGA WGKVGGQAGAHGAE WGKVGGQAGAHGAEA WGKVGGQAGAHGAEALE WGKVGGQAGAHGAEALER WGKVGGQAGAHGAEALERM AEEKEAVLG ARRLGHDFNPDVQAA DAVMGNPKVKAHGK DEVGGEAL DEVGGEALG DEVGGEALGR DEVGGEALGRL DEVGGEALGRLL DGLKHLDN DGLKHLDNL DGLKHLDNLK DGLKHLDNLKG DGLKHLDNLKGTFA DLSNADAV EEKEAVLGL ESFGDLSNA ESFGDLSNADA ESFGDLSNADAV EVGGEALGR EVGGEALGRL FESFGDLSNA FESFGDLSNAD FESFGDLSNADA FESFGDLSNADAV FGDLSNADAV FGDLSNADAVMGNPKVK GDLSNADAV GGEALGRL GGEALGRLL GHDFNPDVQ GHDFNPDVQAA GKVNVDEV GKVNVDEVGGE GKVNVDEVGGEA GKVNVDEVGGEAL GKVNVDEVGGEALG GKVNVDEVGGEALGRL GLWGKVNVDEV HDFNPDVQAA HLSAEEKE HLSAEEKEA HLSAEEKEAV HLSAEEKEAVL HLSAEEKEAVLG HLSAEEKEAVLGL HVDPENF

modification carbamylation

oxidation (M)

oxidation (M)

oxidation (M)

H

obsd (m/z)a

calcd MW (Da)b

charge (+)

396.176 650.857 543.615 543.024 465.699 375.468 399.150 442.173 465.857 546.581 449.190 647.649 473.209 556.261 489.896 395.131 423.647 501.711 558.264 614.819 456.188 512.742 384.827 403.839 510.241 402.637 494.236 470.169 563.214 612.760 444.185 500.740 543.713 601.240 636.765 686.311 504.700 593.622 431.151 386.663 443.215 514.692 585.746 430.180 551.772 587.273 643.825 672.344 538.267 608.308 557.228 471.687 507.214 556.759 613.315 641.829 465.878 429.144

1185.672 1299.715 1627.905 2168.170 929.472 1123.552 1194.589 1323.632 1394.669 1636.796 1792.897 1939.932 944.482 1665.833 1466.766 788.355 845.377 1001.478 1114.562 1227.646 910.451 1023.535 1151.630 1208.651 1527.805 803.366 986.528 938.398 1124.462 1223.531 886.451 999.535 1085.467 1200.494 1271.531 1370.599 1007.456 1777.867 860.388 771.424 884.508 1027.436 1169.510 858.445 1101.530 1172.567 1285.651 1342.673 1611.858 1214.630 1112.489 941.445 1012.483 1111.551 1224.635 1281.656 1394.741 856.372

3 2 3 4 2 3 3 3 3 3 4 3 2 3 3 2 2 2 2 2 2 2 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 3 2

H2O × × ×

100 °C, 1h

after digestiond H2O

100 °C, 1h

× × × ×

× × × × × × × ×

×

×

× × × × × × × × × × × × × × × ×

× × × × × × ×

× × ×

×

× × × × ×

× × × × × × × × × × × × × × ×

× × × × × × × × × × × × × ×

×

× ×

×

× × × × × × × ×


Article


peptide number

peptide sequence

172 173 174 175 176 177 178 179 180 181

HVDPENFR KVNVDEVGGEA KVNVDEVGGEAL KVNVDEVGGEALG LGHDFNPDVQ LGHDFNPDVQAA LSAEEKEAVLG LSAEEKEAVLGL NVDEVGGEA NVDEVGGEAL

182 183 184 185 186 187 188

NVDEVGGEALG NVDEVGGEALGR NVDEVGGEALGRL SAEEKEAVLGL SDGLKHLDN SDGLKHLDNL SDGLKHLDNLK

189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207

SDGLKHLDNLKG SDGLKHLDNLKGTFA SFGDLSNADAV VDEVGGEALGRL VDPENF VDPENFR VGGEALGRL VHLSAEEK VHLSAEEKE VHLSAEEKEAV VHLSAEEKEAVL VHLSAEEKEAVLG VHLSAEEKEAVLGL VNVDEVGGE VNVDEVGGEA VNVDEVGGEAL VNVDEVGGEALG VNVDEVGGEALGRL VVAGVANALAH

208 209 210 211 212

VVYPWTQR VVYPWTQRF WGKVNVDE WGKVNVDEV WGKVNVDEVGGEA

modification

deamidation (N)

dehydration (S)

deamidation (N)

obsd (m/z)a

calcd MW (Da)b

charge (+)

507.210 558.756 615.313 643.827 571.246 642.299 573.287 629.840 445.152 502.195

1012.473 1115.546 1228.630 1285.651 1140.520 1282.594 1144.598 1257.682 888.383 1002.451

2 2 2 2 2 2 2 2 2 2

530.222 608.290 664.843 573.285 499.713 556.264 611.319

1058.488 1214.589 1327.673 1144.598 997.483 1110.567 1220.651

2 2 2 2 2 2 2

× × × ×

432.853 539.258 548.225 607.808 360.604 438.667 436.205 456.699 521.234 606.303 442.197 461.205 498.909 459.212 494.697 551.250 579.767 714.387 511.783

1295.683 1614.837 1094.488 1213.630 719.313 875.414 870.492 911.471 1040.514 1210.619 1323.703 1380.725 1493.809 916.414 987.451 1100.535 1157.556 1426.742 1021.556

3 3 2 2 2 2 2 2 2 2 3 3 3 2 2 2 2 2 2

×

524.752 598.301 473.693 523.238 680.329

1047.550 1194.619 945.456 1044.524 1358.647

2 2 2 2 2

H2O × × × × × × × ×

×

× × × ×

×

× × ×

100 °C, 1h × × × × × ×

after digestiond H2O

100 °C, 1h

×

×

× × × × × × × × × × × × × × × × × × × × × × ×

× ×

×

×

× × × × ×

a

Molecular ion mass observed in the nESI-LC-MS/MS analysis. bCalculated molecular mass in Da of the matched peptide. cPeptides identified at each sample. dPeptides identified at each sample.

inhibitory activities of 7 synthesized peptides (IRLIIVLMPILMA, PAIA, FPAI, ILMA, VFPAIAM, AQILP, and NIGK) isolated from the seaweed Palmaria palmata, NIGK being the most potent peptide with an inhibition value of 50.74% at 1 mg/mL. This is the first work reporting the PAF-AH inhibitory activity of peptides from meat byproducts. Stability of Peptides. Bioactive peptides must undergo processing conditions and gastrointestinal digestion to exert their potential function in vivo. Household preparations including acidic and cooking treatments can destroy amino acids, affect physical and structural properties of proteins and peptides, and modify the digestion rate by gastrointestinal

As can be seen in Figure 4, the inhibitory effect of peptides on PAF-AH activity was low in all of the assayed samples. The control and cooked samples reached PAF-AH inhibitory values between 5.2% and 10.4% before digestion, whereas acidic samples did not show activity. However, the simulated digestion led to an increase of PAF-AH inhibition, mainly for samples H2O and 0.01 N HCl where an approximately 20% increase in PAF-AH inhibitory activities was observed compared to the other analyzed samples. The identification of food-derived peptides showing PAFAH inhibitory activity is very limited. To the best of our knowledge, only Fitzgerald et al.28 reported the PAF-AH I


Article

Journal of Agricultural and Food Chemistry enzymes.29,30 In fact, temperature, pH, amino acid composition, and peptide sequence can affect stability, permeability through gastrointestinal barriers, and solubility of proteins and peptides, leading to a low bioavailability of the biologically active compounds. For instance, ACE-I and DPP-IV inhibitory peptides have been reported to present poor solubility because of their high rate in hydrophobic amino acids, which could require higher amounts of peptide to exert biological effects in the organism.31 Some studies about the stability of bioactive peptides from meat byproducts are available. Fu et al.32 described that ACE inhibitory peptides from bovine collagen hydrolysates kept their activity after temperature, pH treatments, and simulated digestion. This high resistance of collagen peptides could be due to post-translational hydroxylation of the proline residues that are in their sequences.33 On the other hand, a previous work with ham bones showed the release and stability of antioxidant peptides after cooking and digestion, showing that those peptides from collagen gave the differences in the antioxidant activity after digestion and cooking.10 In fact, ACE-I, ECE, DPP-IV, and PAF-AH inhibitory activities found in the present work may be attributed to the previously identified collagen-derived peptides,10 although additional studies are necessary to confirm this statement. Identification of Peptides. Control samples and samples cooked for 1 h were analyzed by nLC-MS/MS to evaluate the peptidic profile before and after digestion, with the distribution of the peptides originating from the main proteins shown in Figure 5. The largest amount of identified peptides in samples before digestion was from hemoglobin protein (alpha and beta subunits), reaching percentages of 53% in the sample H2O and 60% in the sample 100 °C 1 h. After digestion, peptides from collagen protein represented 14% and 21% in the control and cooked sample, respectively, whereas 16% and 19% of the identified peptides were derived from hemoglobin alpha and beta chains. In view of these results, the study was focused on those peptides derived from hemoglobin protein, which is the main constituent of red blood cells and has been previously described as substrate of potential ACE-I, renin, and DPP-IV inhibitory peptides.26 Table 1 shows the sequences of the 212 identified peptides resulting from the alpha and beta subunits of hemoglobin, noting that most of them are present in the samples prior to digestion and are later hydrolyzed by the gastrointestinal enzymes. A sequential loss of amino acids from the C-terminal end is also observed in many of the identified peptides, for instance, in the peptides 1−11 or 16−24 from the hemoglobin alpha. Therefore, cooking and gastrointestinal digestion could give rise to the breakdown of peptides and the generation of smaller fragments that could maintain, increase, or decrease their bioactivity depending on their structure and characteristics. Furthermore, this fact would release a large amount of tripeptides, dipeptides, and free amino acids that could also exert bioactive properties.34 The identified peptides from hemoglobin protein were matched to previously identified ACE-I and DPP-IV inhibitory peptides reported in the BIOPEP database.35 Table 2 shows some examples of dipeptides potentially released from the degradation of the identified peptides that have been described as DPP-IV or ACE inhibitors. Moreover, the peptides 208 and 209 have similar sequences to the peptides VVYPW and VVYPWT, which are porcine hemoglobin peptides previously reported to show ACE-I

Table 2. Dipeptides Potentially Released from the Degradation of the Identified Hemoglobin Peptides Described as DPP-IV or ACE Inhibitors sequence

peptide of origin

AA AD AV DA EA EV GK GL HG NL NV PS RL VH

95, 156, 177 98 198 142 74 144 158 163 119 83, 84, 86 112, 181−184 3, 17 162, 206 68

bioactivity DPP-IV inhibitor, DPP-IV inhibitor DPP-IV inhibitor, ACE inhibitor ACE inhibitor DPP-IV inhibitor, ACE inhibitor DPP-IV inhibitor, ACE inhibitor DPP-IV inhibitor DPP-IV inhibitor DPP-IV inhibitor DPP-IV inhibitor, DPP-IV inhibitor

ACE inhibitor ACE inhibitor

ACE inhibitor ACE inhibitor

ACE inhibitor

ref 15,36 37 37,38 36 39 37,40 39 39,41 39 37 37 37 37,42 37

inhibitory activity.43,44 Some of the identified peptides could be also multifunctional and have a synergistic effect on blood pressure lowering actions,45,46 such as the dipeptides AA, AV, EV, GL, and RL that have been reported to inhibit both ACEI and DPP-IV enzymes.47,48 The great number of short peptides generated during the sample processing could contribute notably to the bioactivities found in the present work. Moreover, di- and tripeptides are more likely to be unaffected to gastrointestinal digestion and to pass directly into the circulatory system.49 In this regard, the use of quantitative methodologies is fundamental in bioavailability studies in order to know the quantity of active peptide that is ingested, absorbed, and able to reach its target site, which will determine its effect and impact in the human system. Nevertheless, the size and low abundance of bioactive peptides as well as the complexity of the food matrix make difficult their quantitation, requiring modern mass spectrometry instruments, bioinformatics tools, and updated protein databases to progress in this field.50 In conclusion, this work shows the potential of dry-cured ham bones as a source of bioactive peptides with in vitro ACE-I, ECE, DPP-IV, and PAF-AH inhibitory activity. The stability of ACE-I and DPP-IV inhibitory peptides was observed after cooking and in vitro digestion of samples. PAF-AH inhibitory activity increased after the action of the gastrointestinal enzymes, while the amount of ECE inhibitory peptides showed a significant decrease. These results suggest that dry-cured ham bones habitually used in the traditional household cooking of stews and broths could have a positive impact on cardiovascular health and a possible reduction of high blood pressure for consumers. However, additional studies would be needed for the identification of the peptide sequences responsible for these bioactivities as well as in vivo studies to assess the bioavailability and physiological effect in the organism.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +34963900022 ext.2114. Fax: +34963636301. E-mail: [email protected]. ORCID

Leticia Mora: 0000-0003-1571-5781 Maria Hayes: 0000-0002-2805-8007 J


Article

Journal of Agricultural and Food Chemistry Funding

(18) Ferrario, C. M.; Strawn, W. B. Role of the renin-angiotensinaldosterone system and proinflammatory mediators in cardiovascular disease. Am. J. Cardiol. 2006, 98, 121−128. (19) Saiga, A. I.; Iwai, K.; Hayakawa, T.; Takahata, Y.; Kitamura, S.; Nishimura, T.; Morimatsu, F. Angiotensin I-converting enzymeinhibitory peptides obtained from chicken collagen hydrolysate. J. Agric. Food Chem. 2008, 56, 9586−9591. (20) Inoue, N.; Hamasaki, A.; Hidaka, S.; Miura, N.; Fukahori, M.; Maruyama, M.; Kawahara, S.; Ohta, K.; Muguruma, M. Analysis of the components of porcine liver hydrolysate and examination of the antioxidant activity and angiotensin converting enzyme (ACE)inhibiting activity. Yakugaku Zasshi 2013, 133, 107−115. (21) Banerjee, P.; Shanthi, C. Isolation of novel bioactive regions from bovine Achilles tendon collagen having angiotensin I-converting enzyme-inhibitory properties. Process Biochem. 2012, 47, 2335−2346. (22) Kedzierski, R. M.; Yanagisawa, M. Endothelin system: the double-edged sword in health and disease. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 851−876. (23) Fernández-Musoles, R.; Salom, J. B.; Martínez-Maqueda, D.; López-Díez, J. J.; Recio, I.; Manzanares, P. Antihypertensive effects of lactoferrin hydrolyzates: Inhibition of angiotensin-and endothelinconverting enzymes. Food Chem. 2013, 139, 994−1000. (24) Drucker, D. J.; Nauck, M. A. The incretin system: glucagonlike peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 2006, 368, 1696−1705. (25) Salles, T. A.; Dos Santos, L.; Barauna, V. G.; Girardi, A. C. Potential role of dipeptidyl peptidase IV in the pathophysiology of heart failure. Int. J. Mol. Sci. 2015, 16, 4226−4249. (26) Lafarga, T.; O’Connor, P.; Hayes, M. Identification of novel dipeptidyl peptidase-IV and angiotensin-I-converting enzyme inhibitory peptides from meat proteins using in silico analysis. Peptides 2014, 59, 53−62. (27) Wilensky, R. L.; Shi, Y.; Mohler, E. R.; Hamamdzic, D.; Burgert, M. E.; Li, J.; Postle, A.; Macphee, C. H. Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development. Nat. Med. 2008, 14, 1059−1066. (28) Fitzgerald, C.; Gallagher, E.; O’Connor, P.; Prieto, J.; MoraSoler, L.; Grealy, M.; Hayes, M. Development of a seaweed derived platelet activating factor acetylhydrolase (PAF-AH) inhibitory hydrolysate, synthesis of inhibitory peptides and assessment of their toxicity using the zebrafish larvae assay. Peptides 2013, 50, 119− 124. (29) Santé-Lhoutellier, V.; Astruc, T.; Marinova, P.; Greve, E.; Gatellier, P. Effect of meat cooking on physicochemical state and in vitro digestibility of myofibrillar proteins. J. Agric. Food Chem. 2008, 56, 1488−1494. (30) Bax, M. L.; Aubry, L.; Ferreira, C.; Daudin, J. D.; Gatellier, P.; Rémond, D.; Santé-Lhoutellier, V. Cooking temperature is a key determinant of in vitro meat protein digestion rate: investigation of underlying mechanisms. J. Agric. Food Chem. 2012, 60, 2569−2576. (31) Lafarga, T.; Hayes, M. Bioactive protein hydrolysates in the functional food ingredient industry: Overcoming current challenges. Food Rev. Int. 2017, 33, 217−246. (32) Fu, Y.; Young, J. F.; Dalsgaard, T. K.; Therkildsen, M. Separation of angiotensin I-converting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature, pH and digestive enzymes. Int. J. Food Sci. Technol. 2015, 50, 1234−1243. (33) Liu, C.; Sugita, K.; Nihei, K. I.; Yoneyama, K.; Tanaka, H. Absorption of hydroxyproline-containing peptides in vascularly perfused rat small intestine in situ. Biosci., Biotechnol., Biochem. 2009, 73, 1741−1747. (34) Sánchez-Rivera, L.; Martínez-Maqueda, D.; Cruz-Huerta, E.; Miralles, B.; Recio, I. Peptidomics for discovery, bioavailability and monitoring of dairy bioactive peptides. Food Res. Int. 2014, 63, 170− 181. (35) Minkiewicz, P.; Dziuba, J.; Iwaniak, A.; Dziuba, M.; Darewicz, M. BIOPEP database and other programs for processing bioactive peptide sequences. J. AOAC Int. 2008, 91, 965−980.

This study was funded by the Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138). A Ramón y Cajal postdoctoral contract to L.M. is acknowledged. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

Proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001.

(1) Udenigwe, C. C.; Aluko, R. E. Food protein-derived bioactive peptides: production, processing, and potential health benefits. J. Food Sci. 2012, 77, R11−R24. (2) Lafarga, T.; Hayes, M. Bioactive peptides from meat muscle and by-products: generation, functionality and application as functional ingredients. Meat Sci. 2014, 98, 227−239. (3) Hartmann, R.; Meisel, H. Food-derived peptides with biological activity: from research to food applications. Curr. Opin. Biotechnol. 2007, 18, 163−169. (4) Ryan, J. T.; Ross, R. P.; Bolton, D.; Fitzgerald, G. F.; Stanton, C. Bioactive peptides from muscle sources: meat and fish. Nutrients 2011, 3, 765−791. (5) Korhonen, H.; Pihlanto-Leppäla, A.; Rantamäki, P.; Tupasela, T. Impact of processing on bioactive proteins and peptides. Trends Food Sci. Technol. 1998, 9, 307−319. (6) Toldrá, F.; Aristoy, M. C.; Mora, L.; Reig, M. Innovations in value-addition of edible meat by-products. Meat Sci. 2012, 92, 290− 296. (7) Toldrá, F.; Mora, L.; Reig, M. New insights into meat byproduct utilization. Meat Sci. 2016, 120, 54−59. (8) Di Bernardini, R.; Harnedy, P.; Bolton, D.; Kerry, J.; O’Neill, E.; Mullen, A. M.; Hayes, M. Antioxidant and antimicrobial peptidic hydrolysates from muscle protein sources and by-products. Food Chem. 2011, 124, 1296−1307. (9) Mora, L.; Reig, M.; Toldrá, F. Bioactive peptides generated from meat industry by-products. Food Res. Int. 2014, 65, 344−349. (10) Gallego, M.; Mora, L.; Hayes, M.; Reig, M.; Toldrá, F. Effect of cooking and in vitro digestion on the antioxidant activity of drycured ham by-products. Food Res. Int. 2017, 97, 296−306. (11) Minkiewicz, P.; Dziuba, J.; Michalska, J. Bovine meat proteins as potential precursors of biologically active peptides-a computational study based on the BIOPEP database. Food Sci. Technol. Int. 2011, 17, 39−45. (12) Bah, C. S.; Bekhit, A. E. D. A.; Carne, A.; McConnell, M. A. Slaughterhouse blood: an emerging source of bioactive compounds. Compr. Rev. Food Sci. Food Saf. 2013, 12, 314−331. (13) Sentandreu, M. A.; Toldrá, F. A rapid, simple and sensitive fluorescence method for the assay of angiotensin-I converting enzyme. Food Chem. 2006, 97, 546−554. (14) Fernández-Musoles, R.; López-Díez, J. J.; Torregrosa, G.; Vallés, S.; Alborch, E.; Manzanares, P.; Salom, J. B. Lactoferricin Bderived peptides with inhibitory effects on ECE-dependent vasoconstriction. Peptides 2010, 31, 1926−1933. (15) Gallego, M.; Aristoy, M. C.; Toldrá, F. Dipeptidyl peptidase IV inhibitory peptides generated in Spanish dry-cured ham. Meat Sci. 2014, 96, 757−761. (16) Erdmann, K.; Cheung, B. W.; Schröder, H. The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. J. Nutr. Biochem. 2008, 19, 643−654. (17) Mora, L.; Hayes, M. Cardioprotective cryptides derived from fish and other food sources: Generation, application, and future markets. J. Agric. Food Chem. 2015, 63, 1319−1331. K


Article

Journal of Agricultural and Food Chemistry (36) Cushman, D. W. Angiotensin converting enzyme inhibitors: Evolution of a new class of antihypertensive drugs. In Mechanisms of Action and Clinical Implications; Horovitz, Z. P., Ed.; Urban & Schwarzenberg: Baltimore, MD, 1981; p 19. (37) Lan, V. T. T.; Ito, K.; Ohno, M.; Motoyama, T.; Ito, S.; Kawarasaki, Y. Analyzing a dipeptide library to identify human dipeptidyl peptidase IV inhibitor. Food Chem. 2015, 175, 66−73. (38) Salampessy, J.; Reddy, N.; Kailasapathy, K.; Phillips, M. Functional and potential therapeutic ACE-inhibitory peptides derived from bromelain hydrolysis of trevally proteins. J. Funct. Foods 2015, 14, 716−725. (39) Cheung, H. S.; Wang, F. L.; Ondetti, M. A.; Sabo, E. F.; Cushman, D. W. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-terminal dipeptide sequence. J. Biol. Chem. 1980, 25, 4015−407. (40) van Platerink, C. J.; Janssen, H. G. M.; Haverkamp, J. Application of at-line two-dimensional liquid chromatography−mass spectrometry for identification of small hydrophilic angiotensin Iinhibiting peptides in milk hydrolysates. Anal. Bioanal. Chem. 2008, 391, 299−307. (41) Nongonierma, A. B.; Mooney, C.; Shields, D. C.; FitzGerald, R. J. Inhibition of dipeptidyl peptidase IV and xanthine oxidase by amino acids and dipeptides. Food Chem. 2013, 141, 644−653. (42) FitzGerald, R. J.; Meisel, H. Lactokinins: Whey protein-derived ACE inhibitory peptides. Nahrung 1999, 43, 165−167. (43) Yu, Y.; Hu, J.; Miyaguchi, Y.; Bai, X.; Du, Y.; Lin, B. Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin. Peptides 2006, 27, 2950− 2956. (44) Ren, Y.; Wan, D. G.; Lu, X. M.; Chen, L.; Zhang, T. E.; Guo, J. L. Isolation and characterization of angiotensin I-converting enzyme inhibitor peptides derived from porcine hemoglobin. Sci. Res. Essays 2011, 6, 6262−6269. (45) Udenigwe, C. C.; Mohan, A. Mechanisms of food proteinderived antihypertensive peptides other than ACE inhibition. J. Funct. Foods 2014, 8, 45−52. (46) Lafarga, T.; Rai, D. K.; O’connor, P.; Hayes, M. Generation of bioactive hydrolysates and peptides from bovine hemoglobin with in vitro renin, angiotensin-I-converting enzyme and dipeptidyl peptidasIV inhibitory activities. J. Food Biochem. 2016, 40, 673−685. (47) Meisel, H.; Walsh, D. J.; Murray, B.; FitzGerald, R. J. ACE inhibitory peptides. In Nutraceutical proteins and peptides in health and disease; Mine, Y.; Shahidi, F., Eds.; CRC Taylor & Francis Group: London, UK, 2006; pp 269−315. (48) Lan, V. T. T.; Ito, K.; Ohno, M.; Motoyama, T.; Ito, S.; Kawarasaki, Y. Analyzing a dipeptide library to identify human dipeptidyl peptidase IV inhibitor. Food Chem. 2015, 175, 66−73. (49) Vermeirssen, V.; Van Camp, J.; Verstraete, W. Bioavailability of angiotensin I converting enzyme inhibitory peptides. Br. J. Nutr. 2004, 92, 357−366. (50) Mora, L.; Gallego, M.; Reig, M.; Toldrá, F. Challenges in the quantitation of naturally generated bioactive peptides in processed meats. Trends Food Sci. Technol. 2017, 69, 306−314.

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