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A Bovine Fibrinogen-Enriched Fraction as a Source of Peptides with in Vitro Renin and Angiotensin-I-Converting Enzyme Inhibitory Activities Tomas Lafarga,† Dilip K. Rai,† Paula O’Connor,‡ and Maria Hayes*,† †

Teagasc, The Irish Agricultural and Food Development Authority, Food BioSciences Department, Ashtown, Dublin 15, Dublin, Ireland ‡ Teagasc, The Irish Agricultural and Food Development Authority, Food BioSciences Department, Moorepark, Fermoy, Co. Cork, Ireland ABSTRACT: Bovine fibrinogen is currently used in the food industry as a binding agent in restructured meat products. However, this protein is underused as a source of bioactive peptides. In this study, a number of novel angiotensin-I-converting enzyme (ACE-I) and renin inhibitory peptides were identified and enriched from a bovine fibrinogen fraction. Fibrinogen was isolated and enriched from bovine blood and hydrolyzed with the food-grade enzyme papain, which was selected for use using in silico analysis. The generated hydrolysate was subjected to ultrafiltration and its peptide profile characterized by liquid chromatography−tandem mass spectrometry. A number of peptides were identified and chemically synthesized to confirm their bioactivity in vitro. Identified peptides included the multifunctional tripeptide SLR, corresponding to f(35−37) of the β-chain of bovine fibrinogen with ACE-I and renin IC50 values of 0.17 and 7.2 mM, respectively. Moreover, the resistance of identified peptides to gastrointestinal degradation and their bitterness were predicted using in silico methods. KEYWORDS: blood, bovine fibrinogen, bioactive peptides, renin, ACE-I, DPP-IV

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INTRODUCTION Bovine slaughterhouse blood represents a valuable protein source, and its use is expected to increase as the world population continues to grow and protein deficiency increases.1 Today, blood proteins are used in the food industry as ingredients, binders, natural color enhancers, emulsifiers, and fat replacers. For example, Fibrimex (Sonac BV, Son, The Netherlands), a combination of the enzyme thrombin (EC 3.4.21.5) and fibrinogen, is currently commercialized as a binding agent.1 However, the use of fibrinogen as a precursor protein for the generation of bioactive peptides has not been investigated to date. The use of bioactive peptides as functional ingredients and pharmaceutical agents has gained much interest in recent years and may provide a commercial opportunity for many companies in addition to improving public health through disease prevention.2 Bioactive peptides are short sequences of amino acids that are inactive within the sequence of the parent protein but have a positive impact on systems of the body once released.3 The use of in silico analysis was recently suggested as the first step in the generation of bioactive peptides from meat muscle and coproduct proteins.4 In silico analysis was previously shown to be an efficient tool for selecting suitable enzymes and for predicting the release of bioactive peptides from known protein sequences.5−7 Peptides with angiotensin-Iconverting enzyme (ACE-I; EC 3.4.15.1), renin (EC 3.4.23.15), and dipeptidyl peptidase-IV (DPP-IV; EC 3.4.14.5) inhibitory properties have been previously generated from a wide variety of natural sources including plant and animal proteins.4,8−10 The inhibition of enzymes involved in the renin−angiotensin− aldosterone system such as ACE-I and renin plays a key role in © 2015 American Chemical Society

the treatment of hypertension, and the inhibition of DPP-IV is a recent approach in the management of type-2 diabetes.11,12 ACE-I acts by cleaving dipeptides from the free C-termini of two typical substrates: angiotensin-I and bradykinin. The cleavage of angiotensin-I results in the generation of angiotensin-II, which stimulates signaling pathways in the heart, blood vessels, kidneys, adipose tissue, pancreas, and brain.11 In addition, ACE-I also degrades bradykinin, a peptide that reduces blood pressure by dilatation of blood vessels.11 Moreover, DPP-IV degrades and inactivates glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP), two incretin hormones that contribute to the enhancement of glucose-induced insulin secretion.12 The aim of this work was to generate and characterize a papain (EC 3.4.22.2) hydrolysate of an enriched bovine fibrinogen protein fraction and to assess its potential as a DPP-IV, ACE-I, and renin inhibitory agent. Papain was the enzyme chosen for hydrolysis of the bovine fibrinogen-enriched protein fraction based on in silico analysis and its use in the meat industry as a tenderizer. The generated papain hydrolysate of the bovine fibrinogen-enriched fraction was then passed through molecular weight cutoff (MWCO) filters (10 and 3 kDa) and further purified by high-performance liquid chromatography (HPLC). A number of peptides were identified using mass spectrometry (MS) and de novo peptide sequencing. ACE-I and renin inhibitory peptides were identified Received: Revised: Accepted: Published: 8676

July 2, 2015 September 15, 2015 September 15, 2015 September 15, 2015 DOI: 10.1021/acs.jafc.5b03167 J. Agric. Food Chem. 2015, 63, 8676−8684

Article

Journal of Agricultural and Food Chemistry

Figure 1. Schematic representation of the procedure followed for the generation of bioactive peptides from bovine fibrinogen. Blood plasma was separated from the cellular fraction by centrifugation at 10000g for 10 min. Plasma was frozen and freeze-dried. A protein fraction rich in fibrinogen was obtained by cold ethanol precipitation, frozen, and freeze-dried. In silico analysis was used for enzyme selection. Papain was chosen as the most efficient enzyme for the generation of a hydrolysate with ACE-I-, renin-, and DPP-I-inhibiting properties. Di- and tripeptides in bold are known ACE-I, renin, or DPP-IV inhibitors available in BIOPEP. Bovine fibrinogen was hydrolyzed using papain, and four fractions were generated by ultrafiltration: FIB-NUFH, FIB-1UFH, FIB-3UFH, and FIB-10UFH. Blood was chilled to 4 °C and handled carefully to minimize hemolysis. Whole blood cells were separated from plasma by centrifugation at 4 °C and 10000g for 10 min using a Sigma 6K10 centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). Plasma was kept at 4 °C, filtered through glass wool, and freeze-dried using an industrial scale freeze-drier, FD 80 model (Cuddon Engineering, Marlborough, New Zealand). The temperature was maintained at 2), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). The bitterness of the synthesized peptides was predicted using the “Q rule” in accordance with a previously published method. Enzymatic Hydrolysis. Papain hydrolysates of the bovine fibrinogen enriched fraction were prepared in triplicate using a BioFlo 110 Modular Benchtop Fermentor (New Brunswick Scientific Co., Cambridge, UK) with agitation, temperature, and pH control. A substrate solution was prepared by resuspending the dried bovine fibrinogen in Milli-Q purified water at a concentration of 10 g/L at a total volume of 500 mL. Agitation, temperature, and pH conditions were adjusted to 350 rpm, 65 °C, and 6.5, respectively. The pH was kept constant using 0.1 M NaOH. Once the optimum pH and temperature conditions were achieved, the enzyme papain (activity ≥ 3 U/mg) was added in a substrate to enzyme ratio of 100:1 (w/w). After 24 h, papain was heat-deactivated at 95 °C for 10 min in a Grant JB Aqua 12 water bath (Grant Instruments, Cambridge, UK). The non-ultrafiltered hydrolysate and the 1, 3, and 10 kDa permeate fractions obtained using MWCO membranes (Millipore, Tullagreen, Carrigtwohill, Co. Cork, Ireland) were labeled FIB-NUFH, FIB1UFH, FIB-3UFH, and FIB-10UFH, respectively. All fractions were frozen, freeze-dried, and stored at −20 °C until further use. Electrophoretic Separation Using Tris-Tricine Peptide Gels. Electrophoresis was carried out using a Bio-Rad Mini-PROTEAN 3 cell System (Bio-Rad Laboratories Ltd., Hertfordshire, UK) and a 10well Mini-PROTEAN Tris-Tricine gel according to the manufacturer’s instructions. Commercial low (1.7−42 kDa in size) and high (2−250 kDa in size) molecular weight (MW) markers were used. Renin Inhibition Assay. This assay was carried out following a previously described method using a renin inhibitor screening assay kit in accordance with the manufacturer’s instructions.5 All fractions were assayed at a concentration of 1 mg sample/mL DMSO in triplicate and standard deviations (SD) calculated. Fluorescence intensity was recorded with a FLUOstar Omega microplate reader (BMG LABTECH GmbH, Offenburg, Germany) using an excitation wavelength of 340 nm and an emission wavelength of 500 nm. The known renin inhibitor Z-Arg-Arg-Pro-Phe-His-Sta-Ile-His-Lys-(Boc)OMe was used as a positive control, and renin IC50 values were determined in triplicate for active peptides and hydrolysates by plotting the percentage of renin inhibition as a function of the concentration of test compound. ACE-I Inhibition Assay. This assay was carried out following a previously described method using an ACE-I inhibitor assay kit and in accordance with the manufacturer’s instructions.5 All fractions were assayed at a concentration of 1 mg/mL HPLC grade water in triplicate, and means and SD were calculated. The known ACE-I inhibitor captopril was used as a positive control at a concentration of 1 mg/mL. Absorbance was measured with a FLUOstar Omega microplate reader (BMG LABTECH GmbH) at 450 nm. ACE-I IC50 values were determined for active hydrolysates and peptides by plotting the percentage of inhibition as a function of the concentration of test compound. DPP-IV Inhibition Assay. This assay was carried out following a previously described method using a DPP-IV inhibitor screening assay kit and in accordance with the manufacturer’s instructions.5 All hydrolysates were assayed in triplicate, and means and SD were calculated. The known DPP-IV inhibitor sitagliptin was used as a positive control. Fluorescence intensity was recorded with a FLUOstar Omega microplate reader (BMG LABTECH GmbH) using an

excitation wavelength of 355 nm and an emission wavelength of 460 nm. DPP-IV IC50 values were determined for active hydrolysates by plotting the percentage of inhibition as a function of the concentration of test compound. De Novo Peptide Sequencing. The FIB-1UFH fraction of the papain hydrolysate of bovine fibrinogen was resuspended in HPLC grade water at a concentration of 1 mg/mL and filtered through a 0.45 μm CHROMAFIL Xtra PVDF-45/25 syringe filter (Macherey-Nagel GmbH & Co., Düren, Germany). The filtered hydrolysate was analyzed using a Q-TOF Premier mass spectrometer (Waters Corp., Milford, MA, USA), coupled to an Alliance 2695 HPLC system (Waters Corp.). The chromatographic separation was carried out at a flow rate of 0.2 mL/min with an injection volume of 10 μL on an Atlantis dC18 column, 100 mm × 2.1 mm, 3 μm particle size (Waters Corp.). Peptides were separated using 0.1% FA in HPLC grade water (solvent A) and 0.1% FA in ACN (solvent B). Column temperature was maintained at 40 °C. The gradient program was as follows: (i) 0 min, 98% A; (ii) 0−0.1 min, 98% A; (iii) 0.1−18 min, 90% A; (iv) 18− 20 min, 85% A; (v) 20−21 min, 40% A; (vi) 21−22 min, 20% A; (vii) 22−25 min, 98% A; and (viii) 25−30 min, 98% A. HPLC-MS/MS was performed using a data-dependent acquisition (DDA) on electrospray ionization (ESI) in positive ion mode at 1 s scan. Argon was used as collision gas with the collision energy ramp from 15 eV for low MW peptides to 60 eV for high MW peptides. The tandem mass spectrometry (MS/MS) spectral data were deconvoluted using the MaxEnt 3 algorithm, and their amino acid sequences were determined using the peptide sequencing software available in the Waters Biolynx software package. Chemical Synthesis. Peptides were synthesized by microwaveassisted solid phase peptide synthesis (MW-SPPS) performed on a Liberty CEM microwave peptide synthesizer (Mathews, NC, USA). Peptides were synthesized on H-Ala-HMPB-ChemMatrix and H-IleHMPB-ChemMatrix resins (PCAS Biomatrix Inc., Quebec, Canada) and purified using RP-HPLC on a Semi Preparative Jupiter Proteo (4u, 90A) column (Phenomenex, Cheshire, UK). Fractions containing the desired molecular mass were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) and were pooled and freeze-dried on a Genevac HT 4X lyophilizer (Genevac Ltd., Ipswich, UK). Statistical Analysis. All tests were replicated three times, and mean values and SD were calculated. ANOVA one-way analysis was carried out using Minitab v17 (Minitab Ltd., UK). When significant differences were present, a Tukey pairwise comparison of the means was conducted to identify where the sample differences occurred.

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RESULTS AND DISCUSSION In Silico Analysis. In silico analysis was previously shown to be an efficient method to predict the release of bioactive peptides with prolyl endopeptidase- (PEP; EC 3.4.21.26), ACEI-, and DPP-IV-inhibiting properties.5−7,16 In this work, a bovine fibrinogen enriched protein fraction was generated and hydrolyzed using papain to generate bioactive peptides with ACE-I, renin, and DPP-IV inhibitory activities. Bovine fibrinogen is composed of two sets of disulfide-bridged α-, β-, and γ-chains.17 The amino acid sequences of bovine fibrinogen α-, β-, and γ-chains were accessed from the UniProt database. In silico analysis was carried out using BIOPEP to select a suitable enzyme to generate a hydrolysate rich in bioactive peptides with potential to inhibit ACE-I, renin, and DPP-IV (Figure 1). A number of enzymes including papain, pepsin, trypsin, ficain, bromelain, and thermolysin were trialled using in silico analysis to generate peptides from bovine fibrinogen. Table 1 describes the number of known ACE-I-, renin-, and DPP-IV-inhibiting peptides, available in BIOPEP, which were generated in this study from bovine fibrinogen using selected enzymes in silico. Few renin inhibitory peptides were predicted to be released from bovine fibrinogen by in silico cleavage 8678

DOI: 10.1021/acs.jafc.5b03167 J. Agric. Food Chem. 2015, 63, 8676−8684

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compared to ACE-I and DPP-IV inhibitory peptides. This may be due to the limited reports that exist regarding the generation of renin inhibitors from natural sources. Renin and DPP-IV inhibitory peptides generated in silico include the peptides IR and FA, corresponding to f(180−181) and f(533−534) of the α-chain of bovine fibrinogen. The peptide FA was previously reported to inhibit DPP-IV,18 and the peptide IR was previously generated from pea protein and was reported to have a renin IC50 value of 9.2 ± 0.2 mM.19 Papain was the enzyme selected for use in vitro in this study. Papain was used previously to generate ACE-I and renin inhibitors as well as antioxidant peptides from seaweed as well as bovine proteins.20,21 Moreover, papain was found to generate the largest number of known ACE-I- and renin-inhibiting peptides from fibrinogen. In addition, application of papain to bovine fibrinogen generated 48 DPP-IV-inhibiting peptides. This compared favorably to the enzymes pepsin, trypsin, bromelain,

Table 1. Number of ACE-I, Renin, and DPP-IV Inhibitory Sequences Found within Bovine Fibrinogen after in Silico Hydrolysis with BIOPEP no. of known bioactive peptides enzyme used for in silico hydrolysis

ACE-I inhibitorsa

renin inhibitorsa

DPP-IV inhibitorsa

papain (EC 3.4.22.2) pepsin (EC 3.4.23.1) trypsin (EC 3.4.21.4) bromelain (EC 3.4.22.4) thermolysin (EC 3.4.24.27) ficain (EC 3.4.22.3)

72 3 5 45 28 58

3 0 2 0 1 0

48 8 4 22 22 61

a Data accessed from BIOPEP, available at http://www.uwm.edu.pl/ biochemia/index.php/pl/biopep on April 2015.

Figure 2. Tris-tricine peptide gel of the isolated fibrinogen protein and fractions generated by hydrolysis and ultrafiltration. Low and high MW markers are shown in columns (a) and (g), respectively. The isolated fibrinogen-rich fraction is shown in column ( f). FIB-1UFH, FIB-3UFH, FIB10UFH, and FIB-NUFH are shown in columns (b), (c), (d), and (e), respectively. 8679


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Journal of Agricultural and Food Chemistry and thermolysin, which generated 8, 4, 22, and 22 DPP-IV inhibitory peptides, respectively, following in silico analysis (Table 1). Generation of a Bovine Fibrinogen-Enriched Protein Fraction. Plasma constitutes approximately 65−70% of the total weight of blood and can be separated from the cellular fraction by centrifugation.22 Yields for cellular and plasma fractions were calculated as 61.65 ± 0.31 and 38.35 ± 0.32% for the plasma and the cellular fraction, respectively. Moreover, the yield of the fibrinogen-rich fraction was found to be 1.5 ± 0.2 g/L. This fraction was analyzed by electrophoresis using a TrisTricine 10−20% peptide gel. Results, shown in Figure 2, suggest that although some impurities were present in the isolated fraction, fibrinogen was the main constituent with bands shown at 63.5 kDa (α-chain), 56 kDa (β-chain), and 47 kDa (γ-chain). In addition, the total protein content of the fibrinogen-rich fraction generated following the procedure described in Figure 1 was determined as 87.9 ± 0.3% using the LECO method. This value is comparable to the protein contents obtained in previous hydrolysis studies where high protein yields were obtained following hydrolysis of peanut.23 In addition, the use of fibrinogen in combination with the enzyme thrombin was determined as safe for human consumption by the European Food Safety Authority (EFSA) in 2005.24 Bovine blood proteins including hemoglobin and serum albumin have previously been used for the generation of bioactive proteins,1,4 but to the best of our knowledge, no ACEI, renin, or DPP-IV inhibitors have been generated from bovine fibrinogen to date. The identification of bioactive peptides within the sequence of bovine fibrinogen adds further value to this protein product. Electrophoresis results, shown in Figure 2, demonstrate that the generated fibrinogen protein fraction was hydrolyzed by papain and that the fractions generated by MWCO filtration consist mainly of peptides with MW 50% compared to the positive control captopril, which had an ACE-I inhibitory activity of 88.10 ± 4.75%. The ACE-I IC50 values of FIB-NUFH, FIB-1UFH, FIB-3UFH, and FIB-10UFH were calculated and determined as 1.86 ± 0.12, 0.92 ± 0.08, 1.41 ± 0.09, and 1.55 ± 0.09 mg/mL, respectively. Ultrafiltration of FIB-NUFH led to increased ACE-I inhibitory activity, and the FIB-1UFH fraction had the lowest ACE-I IC50 value compared to the FIB-NUFH, FIB-10UFH, and FIB3UFH fractions (p < 0.05). These results are consistent with the fact that peptidic ACE-I inhibitors usually consist of short amino acid sequences.25 Results are comparable to inhibition values obtained for other protein hydrolysates, where sequential ultrafiltration of blood protein hydrolysates with 10, 3, and 1 kDa membranes resulted in increased ACE-I-inhibiting activity.26 Furthermore, the renin inhibitory activity of the generated hydrolysates was measured at a concentration of 1 mg/mL. Again, MWCO led to a significant increase in the renininhibiting activity of the FIB-1UFH fraction compared to the FIB-NUFH, FIB-10UFH, and FIB-3UFH fractions (p < 0.05). These results are consistent with the fact that dipeptides were

Figure 3. ACE-I-, renin-, and DPP-IV-inhibiting properties in vitro of studied papain hydrolysates and ACE-I, renin, and DPP-IV inhibitory bioactivities of papain hydrolysates of bovine fibrinogen when tested in vitro at a sample concentration of 1 mg/mL. Z-Arg-Arg-Pro-Phe-HisSta-Ile-His-Lys-(Boc)-OMe, sitagliptin, and captopril were used as positive controls for renin, DPP-IV, and ACE-I, respectively. Enzyme inhibition is expressed as percent inhibition, and the values represent the means of three independent experiments ± SD. For each bioactivity tested, bars with different letters have mean values that are significantly different (p < 0.05).

previously suggested as the most effective for renin inhibition.19 Furthermore, long peptides present within a hydrolysate can mask the activity of small active peptides. The activity of the FIB-10UFH fraction was lower than that of FIB-NUFH (p < 0.05). This may be due to the presence of one or more polypeptides with renin inhibitory properties within the FIBNUFH fraction, as longer peptides such as IRLIIVLMPILMA have been reported to inhibit renin.21 The fraction FIB-1UFH was the most active and inhibited renin by 32.09 ± 1.93%. This result concurs with previously reported research on flaxseed protein hydrolysates and renin inhibitory activities.27 In this work, flaxseed was hydrolyzed with Thermoase and only a marginal improvement in renin inhibitory activity was observed for virtually all samples after membrane ultrafiltration.27 No studies regarding the use of blood proteins for the generation of renin-inhibiting hydrolysates and peptides have been carried out to date. In Vitro DPP-IV Inhibitory Activity. The in vitro DPP-IV inhibitory activity of the studied fractions was calculated, and results are shown in Figure 3. All studied fractions showed a moderate inhibition of DPP-IV. The most active fraction, FIB3UFH, inhibited DPP-IV by 41.29 ± 1.17% when tested at a concentration of 1 mg/mL. The DPP-IV IC50 value of this fraction was calculated and was 1.03 ± 0.01 mg/mL. A slight increase in the DPP-IV-inhibiting activity of the ultrafiltered fractions compared to the nonultrafiltered fraction was observed (p < 0.005). The removal of higher MW fractions previously led to an increase in the DPP-IV inhibitory activity in vitro.28 Moreover, most of the peptides with DPP-IV inhibitory activity described in the literature to date are between two and eight amino acid residues in length.7 The DPP-IV-inhibiting activities of the papain hydrolysates generated in this study were comparable to those of other hydrolysates from natural sources. These include the trypsin hydrolysates of Amaranthus hypochondriacus L. proteins, with IC50 values ranging from 1.2 to 2.0 mg/mL, depending on the enzyme to substrate ratio,28 and a Flavourzyme hydrolysate of Atlantic salmon gelatin with an IC50 value of 1.35 mg/mL.29 The IC50 values obtained herein were also similar to those 8680


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Figure 4. ESI-MS/MS spectra for de novo sequence determination of peptides KR and KYK. Peptides KR, FR, YF, RR, YR, SLK, SPR, and KYK were identified by de novo peptide sequencing software within the BioLynx software package (Waters Corp., Milford, MA, USA).

available in BIOPEP, as shown in Table 2. For example, the peptide RR, corresponding to f(145−147) and f(452−453) of the α-chain of bovine fibrinogen, was found to share the same peptides at the C-terminus of the peptide with the previously reported DPP-IV inhibitor WRR, which had a reported DPP-IV IC50 value of 570 ± 62 μM.33 Chemical Synthesis and Confirmation of Bioactivity. A number of the identified peptides were selected for chemical synthesis and bioassay assessment. PeptideRanker scores were calculated for each peptide and are shown in Table 2. Peptide selection for chemical synthesis was based on the current knowledge of bioactive peptides, the known attributes of ACE-I and renin inhibitors, and the score assigned by PeptideRanker to each peptide (Table 2). The amino acids tryptophan, tyrosine, proline, and phenylalanine were reported previously as the most effective amino acids in an ACE-I-inhibiting peptide when they are present at the C-terminal side of a dipeptide.34 A quantitative structure−activity relationships study (QSAR), which modeled biological activity of ACE-I-inhibiting peptides as a function of molecular structure, suggested that amino acid residues with large bulky chains as well as hydrophobic side chains such as found in the amino acids phenylalanine, tryptophan, and tyrosine are the most effective residues in a dipeptide.25 Amino acid residues with small as well as hydrophobic side chains, which include valine, leucine, and isoleucine, were suggested for the N-terminal side of ACE-I inhibitory peptides. Hydrophobic amino acid residues with high

obtained from a trypsin hydrolysate of whey protein, which had an IC50 value of 1.51 mg/mL. The pentapeptide IPAVF, corresponding to β-lactoglobulin f(78−82), had an IC50 value of 44.7 μM and was responsible for the observed DPP-IV inhibitory activity.30 Peptide Identification by de Novo Peptide Sequencing. The characterization of bioactive peptides within an active protein hydrolysate is of key importance. To identify bioactive peptides, it is often necessary to fractionate and purify the whole hydrolysate using filtration and chromatography methods. Today, widely used methods for peptide fractionation and enrichment include ultrafiltration and liquid chromatography.31 In this study, the identification of peptides contained in FIB-1UFH was carried out using HPLC-MS/MS. A total of 20 di-, tri-, and tetrapeptides were identified by de novo peptide sequencing as represented in Figure 4. These peptides are listed in Table 2 together with their position in the parent protein and their observed masses as well as their calculated masses. Identified bioactive peptides were compared to previously reported ACE-I, DPP-IV, and renin inhibitors available in the database BIOPEP.14 A number of the identified peptides were bioactive and were previously generated from various natural sources. For example, the peptide RR was generated by the action of dipeptidyl peptidases purified from porcine skeletal muscle and was reported to inhibit ACE-I.32 There were sequence similarities between the peptides identified in this study and previously reported ACE-I and DPP-IV inhibitors 8681


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Journal of Agricultural and Food Chemistry Table 2. Peptides Identified from Bovine Fibrinogen by HPLC-MS/MS amino acid sequence

parent protein

obtained mass charge (Da)

calcd mass (Da)

PeptideRanker scorea

similar ACE-I-inhibiting peptidesb KR, FQKPKR, IPALLKR FR, WTFR, AFKDEDTEEVPFR YFYPEL RR, YLYEIARR YRPY ALKAWSVAR, YLYEIAR LYPVK, VK, AFDAVGVK, AHEPVK LYPVK, VK, AFDAVGVK, AHEPVK LYPVK, VK, AFDAVGVK, AHEPVK YPR, PR, IYPR, WNPR, QLGFLGPR N/A ALKAWSVAR, YLYEIAR LDAQSAPLR ALKAWSVAR, YLYEIAR LDAQSAPLR NMAINPSK LDAQSAPLR N/A VLDTDYK, VLAQYK, GYK, IYK, YK LYPVK, VK, AFDAVGVK, AHEPVK

KR

f(170−171) FIBA and f(219−220) FIBG

302.17

302.20

0.21

FR

f(388−389; 408−409) FIBA

321.17

321.18

0.99

YF RR YR PAR

f(464−465) FIBB and f(191−192; 305−306) FIBG f(145−146; 452−453) FIBA f(48−49; 243−244) FIBB and f(299−300) FIBG f(599−601) FIBA

327.87 330.17 337.17 342.17

328.14 330.21 337.17 342.20

0.98 0.57 0.53 0.60

VVK

f(476−478) FIBA

344.27

344.24

0.03

TVK

f(163−165) FIBB and f(166−168) FIBG

346.17

346.22

0.03

LVK

f(85−87) FIBG

358.17

358.25

0.07

SPR

f(288−290) FIBA

358.17

358.19

0.62

VSR KAR

f(114−146) FIBB f(596−598) FIBA

360.27 373.28

360.21 373.24

0.09 0.17

SLR EAR

f(35−37) FIBB f(80−82) FIBG

374.28 374.18

374.22 374.19

0.40 0.09

VLR YSK NLR DSSD KYK

f(174−176) f(379−381) f(181−183) f(323−326) f(351−353)

386.28 396.28 401.28 421.98 437.28

386.26 396.20 401.23 422.12 437.26

0.20 0.13 0.34 0.10 0.11

EVVK

f(475−478) FIBA

473.28

473.28

0.02

FIBB and f(270−272) FIBG FIBG FIBB and f(147−149) FIBG FIBG FIBB

similar DPP-IVinhibiting peptidesb KR FR, EQLTKCEVFR YF WRR RR, WRR PACGGFYISGRPG VKSVVAL, VK

VKSVVAL, VK

LVSGM, VKSVVAL, VK SP N/A N/A SL N/A VLGP, VLVLDTDYK N/A N/A N/A VLVLDTDYK EVTFPA

a

Data accessed from PeptideRanker, available at http://bioware.ucd.ie/ in April 2015. bData accessed from BIOPEP, available at http://www.uwm. edu.pl/biochemia/index.php/pl/biopep in April 2015.

inhibited the activity of ACE-I by half at a concentration of 366, 518, and 1095 μM, respectively.35 Selected peptides were also screened for their ability to inhibit renin. Only the dipeptide YR and the tripeptide SLR were found to inhibit renin with IC50 values of 8.78 ± 0.37 and 7.29 ± 0.16 mM, respectively. These results compare favorably to previously describe renin inhibitors including the peptides IR and IRLIIVLMPILMA, generated by enzymatic hydrolysis of pea and seaweed proteins and with calculated IC50 values of 9.2 and 3.3 mM, respectively.19,21 Prediction of Resistance to Gastrointestinal Degradation and Bitterness. The use of bioactive peptides as functional ingredients is usually limited by a number of issues including low stability of the peptides against gastrointestinal degradation. Although bioavailability should be assessed in vivo, resistance of a peptide to degradation can be predicted by computer simulations of proteolysis. In this study, the in silico tool ExPASy PeptideCutter was used to predict the stability of the identified bioactive peptides after a simulated gastrointestinal digestion. Results suggest that the peptide RR may be resistant to degradation by pepsin, trypsin, and chymotrypsin. The peptides FR, YR, and YF were predicted to be cleaved by both pepsin and chymotrypsin into the amino acids phenylalanine and arginine, tyrosine and arginine, and tyrosine and phenylalanine, respectively. Moreover, the peptide KR was

electronic properties such as proline, phenylalanine, and tryptophan are usually found at the C-terminal end of active ACE-I inhibitory tripeptides.25 Furthermore, dipeptides with hydrophobic residues at the N-terminus and a bulky or aromatic group at the C-terminus were observed to be the most effective peptides for renin inhibition.19 The dipeptides KR, FR, YF, RR, and YR and the tripeptides SLR and KYK were chemically synthesized by MW-SPPS and tested for ACE-I- and renin-inhibiting properties in vitro. The IC50 values were determined for active peptides by plotting the percentage of enzyme inhibition as a function of the concentration of test compound. At a concentration of 1 mg/ mL, the peptides KR, FR, YF, YR, SLR, and KYK inhibited ACE-I by >60%. ACE-I IC50 values were calculated for active peptides and were found to be 383.76 ± 1.64, 425.12 ± 21.49, 94.62 ± 5.83, 124.12 ± 8.40, 171.95 ± 25.44, and 280.12 ± 33.14 μM for KR, FR, YF, YR, SLR, and KYK, respectively. The peptide RR is a known ACE-I inhibitor with an IC50 value of 267.1 ± 11.3 μM.32 No ACE-I peptide inhibitors have been generated from bovine fibrinogen to date. However, the ACE-Iinhibiting activity of the peptides identified in this study compared favorably to ACE-I-inhibiting peptides generated from bovine blood proteins such as hemoglobin.35 These include the peptides corresponding to f(67−106), f(73−105), and f(100−105) of the α-chain of bovine hemoglobin, which 8682



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predicted to be degraded by trypsin, and the tripeptides SLR and KYK were predicted to be broken down into the dipeptides SL and LR and KY and YK, respectively. The peptides SL and KY are known DPP-IV inhibitors, and the peptides KY and YK were both previously reported to inhibit ACE-I and are available in BIOPEP. Bioactive hydrolysates and peptides are usually bitter in taste, and Western consumers do not seem to be willing to compromise taste for health benefits.36 A number of methods have been used to predict the bitterness of a peptide. The “Q rule” formulated by Ney37 quantifies the bitterness of a peptide on the basis of its amino acid composition, whereby a Q value is calculated from the solubility data of the individual amino acids. According to this method, when the Q value of a peptide, with a MW under 6 kDa, exceeds 1400 cal/mol, this peptide will be almost certainly bitter. The Q values for the peptides KR, FR, YF, RR, YR, SLR, and KYK were 1125.0, 1337.0, 2400.0, 750.0, 1525.0, 950.0, and 1766.6 cal/mol, respectively. Obtained Q values suggest that the peptides YF, YR, and KYK may be bitter in taste. Moreover, the Q value of peptides generated by the action of digestive enzymes in silico was also calculated. The peptides SL, LR, KY, and YK, which were predicted to be generated by the cleavage of the tripeptides SLR and KYK, had Q values of 1050.0, 1275.0, 1900.0, and 1900.0 cal/mol, respectively. The peptide KYK was predicted to be degraded by gastrointestinal enzymes and to be cleaved into the peptides KY and YK, which had higher predicted Q values and therefore could have enhanced bitterness following digestion. Ney’s rule can be applied to the majority of known peptides, but there are exceptions. For example, a recent study characterized the flavor, chemical properties, and hydrophobicity of a number of peptides derived from two commercial enzymatic hydrolysates of soy protein. The obtained hydrophobicity data based on Q values did not support Ney’s rule as a predictor of bitterness.38 In this work a sequential fractionation and enrichment methodology was used to generate fibrinogen-enriched fractions with inhibitory activities against the enzymes ACE-I, DPP-IV, and renin. This is the first report that evaluates the ACE-I, renin, and DPP-IV inhibitory activity from papain hydrolysates of bovine fibrinogen. Results presented in this study show the presence of biologically active peptides with renin- and ACE-I-inhibiting properties. These peptides were encrypted in bovine fibrinogen and released by enzymatic cleavage with the food-grade enzyme papain. The generated papain hydrolysates and peptides were found to inhibit renin, ACE-I, and DPP-IV, and the percentage inhibition values obtained compared favorably with those found previously for hydrolysates and peptides isolated from animal and plant sources. A number of known as well as novel bioactive peptides were generated and identified. The peptides identified herein were predicted to be bitter and to be degraded by digestive enzymes in silico. However, in silico analysis is not enough to predict the resistance of a peptide to degradation and its taste, and in vivo studies in animal models are required to assess the bioavailability of a peptide after ingestion. These results demonstrate the potential of bovine fibrinogen as a resource for bioactive peptide generation and opens new commercial opportunities for its use beyond its current applications in the food industry.

Article

AUTHOR INFORMATION

Corresponding Author

*(M.H.) Phone: +353 (0) 1 8059957. E-mail: maria.hayes@ teagasc.ie. Funding

T.L. is in receipt of a Teagasc Walsh Fellowship. This work forms part of the ReValueProtein Research Project (Grant Award 11/F/043), which is supported by the Irish Department of Agriculture, Food and the Marine (DAFM) and the Food Institutional Research Measure (FIRM), both funded by the Irish government under National Development Plan 20072013. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED 1UFH, hydrolysate ultrafiltrated with a 1 kDa membrane; 3UFH, hydrolysate ultrafiltered with a 3 kDa membrane; 10UFH, hydrolysate ultrafiltered with a 10 kDa membrane; ACE-I, angiotensin-I-converting enzyme; ACN, acetonitrile; DDA, data-dependent acquisition; DMSO, dimethyl sulfoxide; DPP-IV, dipeptidyl peptidase-IV; EFSA, European Food Safety Authority; ESI, electrospray ionization; FA, formic acid; GIP, gastric inhibitory peptide; GLP-1, glucagon-like peptide-1; HPLC, high-performance liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MW, molecular weight; MWCO, molecular weight cutoff; MW-SPPS, microwave-assisted solid phase peptide synthesis; NUFH, nonultrafiltered hydrolysate; PEP, prolyl endopeptidase; SD, standard deviation; TOF, time-of-flight; QSAR, quantitative structure−activity relationship

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REFERENCES

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