Addition of an Enzymatic Hydrolysate of Bovine Globulins to Bread

Article pubs.acs.org/JAFC

Addition of an Enzymatic Hydrolysate of Bovine Globulins to Bread and Determination of Hypotensive Effects in Spontaneously Hypertensive Rats Tomas Lafarga,† Eimear Gallagher,‡ Rotimi E. Aluko,§ Mark A. E. Auty,# and Maria Hayes*,† †

Food BioSciences Department, Teagasc, The Irish Agricultural and Food Development Authority, Ashtown, Dublin 15, Dublin, Ireland ‡ Food Chemistry Department, Teagasc, The Irish Agricultural and Food Development Authority, Ashtown, Dublin 15, Dublin, Ireland § University of Manitoba, Department of Human Nutritional Sciences, Winnipeg R3T 2N2, Canada # Food Chemistry Department, Teagasc, The Irish Agricultural and Food Development Authority, Moorepark, Fermoy, Co. Cork, Ireland ABSTRACT: The aim of this study was to develop bread containing a papain hydrolysate of bovine α- and β-globulins (GPH) with in vitro and in vivo antihypertensive activities. The physical characteristics of the formulated bread were assessed over a six day period and results suggested that the overall quality and acceptance of bread was not affected by the inclusion of GPH at a concentration of 4% (w/w). Bright field light microscopy and confocal scanning laser microscopy images were used to visualize the main ingredients of the bread. In addition, the antihypertensive activity of the bread was assessed in spontaneously hypertensive rats (SHRs) over a 24 h period where a maximum significant decrease in systolic blood pressure of 36.2 ± 1.9 mmHg was observed 8 h after oral administration. Results demonstrate that the antihypertensive activity of GPH was resistant to the baking process and shows potential for use as a functional antihypertensive ingredient. KEYWORDS: hypertension, functional foods, spontaneously hypertensive rats, ACE-I, bioactive peptides

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INTRODUCTION Cardiovascular diseases are the leading cause of death and disability-adjusted life years lost.1 It is estimated that, by 2030, over 23.3 million people will die annually from cardiovascular diseases.2 Approximately half of the global burden of cardiovascular diseases can be attributed to hypertension or high blood pressure,3 which is usually defined by the presence of a chronic increase in systemic arterial pressure above a certain threshold value.4 The World Health Organization (WHO) classifies hypertension in three subgroups: (i) grade 1 or mild hypertension, (i) grade 2 or moderate hypertension, and (iii) grade 3 or severe hypertension. Mild hypertension was defined for subjects with systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels in the ranges 140−159 and 90−99 mmHg, respectively.5 However, according to the European Society of Hypertension (ESH), the real threshold for hypertension must be considered flexible, depending on the total cardiovascular risk profile of each individual.6 Inhibition of enzymes including angiotensin-I-converting enzyme (ACE-I; EC 3.4.15.1) and renin (EC 3.4.23.15) within the renin-angiotensin-aldosterone system (RAAS) plays a key role in the treatment of hypertension. Indeed, a common pharmacological strategy for the treatment of hypertension is the prescription of ACE-I and renin inhibitory drugs, including captopril, enalapril, and aliskiren. Furthermore, ACE-I and renin inhibitory peptides were previously generated from natural sources, such as fish,7,8 pulses,9 and dairy,10,11 and coproducts from the meat processing industry, including blood.12,13 Although numerous bioactive hydrolysates and © 2016 American Chemical Society

peptides with in vitro ACE-I and renin inhibitory properties have been generated to date, only a small number of these ingredients have been assessed in animal models and have demonstrated antihypertensive effects in vivo. Examples include the commercially available products Calpis sour milk, containing the peptides IPP and VPP, and Valtyron, which contains the dipeptide VY. These products were repeatedly found to have blood pressure lowering effects when orally administered to hypertensive patients. 14−18 In Europe, following an application from Valio Ltd. submitted pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Finland, the Panel on Dietetic Products, Nutrition, and Allergies of the European Food Safety Authority (EFSA) concluded that a cause and effect relationship between the consumption of IPP and VPP and maintenance of normal blood pressure has not yet been established.19 However, despite the lack of a proven cause and effect relationship, bioactive hydrolysates and peptides show potential for use as ingredients in functional foods. Functional foods can play a key role in the maintenance of health and are defined as foods that impart a health benefit above and beyond basic nutrition.20 Foods ideally suited for use as bioactive delivery vehicles include bread as it is widely, easily, and regularly Received: Revised: Accepted: Published: 1741

December 30, 2015 February 11, 2016 February 13, 2016 February 13, 2016 DOI: 10.1021/acs.jafc.5b06078 J. Agric. Food Chem. 2016, 64, 1741−1750

Article

Journal of Agricultural and Food Chemistry

of the pH to 7.2. A second fraction (labeled as FII), rich in γ-globulins, was recovered by addition of ethanol to the plasma solution at a final concentration of 19% (v/v). The pH was maintained at 7.2. A third protein fraction (FIII or GPH), rich in α- and β-globulins, was obtained by adjusting the pH of plasma to 5.5 and subsequent addition of ethanol to a final concentration of 40% (v/v). Ethanol was added drop by drop, and the process was carried out at temperatures close to 0 °C to minimize protein denaturation. After each precipitation step, proteins were separated by centrifugation at 4 °C and 10,000g for 5 min. GPH was resuspended in water, frozen, and freeze-dried. Enzymatic Hydrolysis of Bovine Blood Fractions. Papain hydrolysates of FIII were prepared in triplicate using a BioFlo 110 Modular Benchtop Fermentor (New Brunswick Scientific Co., Cambridge, England, UK) with agitation and controlled temperature and pH. A substrate solution was prepared by suspending the dried protein fraction in Milli-Q purified water at a concentration of 15 g/L at a total volume of 500 mL. Temperature, agitation, and pH conditions were adjusted to 65 °C, 350 rpm, and 6.5, respectively. The pH was kept constant using 0.1 M NaOH. Once the optimum conditions were achieved, the enzyme papain was added in a substrateto-enzyme ratio of 100:1 (w/w). After 24 h, papain was heatdeactivated at 95 °C for 10 min. The generated dried globulin protein hydrolysate was labeled as GPH. Peptide Identification by LC-MS/MS. The peptidic content of the generated papain hydrolysate was analyzed using a Thermo Scientific Q Exactive mass spectrometer connected to a Dionex UltiMate 3000 RSLCnano LC System. The samples were suspended in 0.1% FA in HPLC grade water and cleaned using Millipore C18 ZipTips prior to LC-MS/MS. The sample was loaded onto a Biobasic Picotip Emitter (120 mm length, 75 μm ID) packed with Reprocil Pur C18 (1.9 μm) reversed phase media and was separated by an increasing ACN gradient over 60 min at a flow rate of 250 nL/min. The mass spectrometer was operated in positive ion mode with a capillary temperature of 220 °C and with a potential of 2.3 kV applied to the emitter. All data were acquired with the mass spectrometer operating in automatic data-dependent switching mode. A high resolution (70,000 fwhm) MS scan (m/z 300−1600) was performed using the Q-Exactive to select the 12 most intense ions prior to MS/ MS analysis using higher-energy collisional dissociation. The raw data was de novo sequenced and searched against the bovine subset of the UniProtKB/Swiss-Prot database using the search engine of PEAKS Studio 7 for peptides cleaved with no specific enzyme. At least one unique peptide was required to identify a protein. Each peptide used for protein identification met specific PEAKS parameters (only peptide scores that corresponded to a false discovery rate of ≤1% were accepted from the PEAKS database search). Each sample was run three times, and the results shown are the combined technical replicates. Peptide sequences, their position inside the parent proteins, their observed masses, and their retention times were provided by PEAKS Studio software. The potential of the identified peptides to be bioactive was assessed using PeptideRanker, available at http://bioware.ucd.ie.30 Renin Inhibition Assay. This assay was carried out using a renin inhibitor screening assay kit in accordance with the manufacturers’ instructions. All samples were assayed at a concentration of 1 mg of sample/mL of DMSO in triplicate and standard deviations (S.D.) were 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-PheHis-Sta-Ile-His-Lys-(Boc)-OMe was used as a positive control, and renin IC50 values were determined in triplicate for active 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 using an ACE-I inhibitor assay kit in accordance with the manufacturers’ instructions. All fractions were assayed at a concentration of 1 mg of sample/mL of 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

consumed and has previously been used as a vehicle for the delivery of chitosan,21 omega-3 fatty acids,22 and folic acid.23 In addition, bread was recently used as a food vehicle for the delivery of a protein hydrolysate with renin inhibitory activity.24 The authors demonstrated by confocal scanning laser microscopy and in vitro bioassays that the renin inhibitory activity of the hydrolysate was not altered during the baking process. The use of functional food ingredients is becoming more important in the baking industry,25 and a number of functional breads have been developed, including a bread containing grape seed with increased antioxidant activity and gallic acid and catechin content.26 Nutritional improvements of wheat bread have also been achieved by adding pasteurized sweet cheese whey solids,27 and even fish protein concentrates have been used as a source of proteins for increasing the lysine content in wheat breads.28 The aim of this work was to examine the sensory, physical, and bioactive properties of wheat bread formulated using an enzymatic hydrolysate of bovine α- and β-globulins (GPH) compared to a negative control (white bread) and bread containing a commercially available whey protein hydrolysate (WPH). The effect of the inclusion of GPH and WPH on parameters including volume, color, texture, moisture, and crumb grain structure were studied over a six day period and compared to the negative control bread. Moreover, the antihypertensive activity of the generated hydrolysate and bread formulated with GPH was assessed in vivo using spontaneously hypertensive rats (SHRs) over a 24 h period.

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MATERIALS AND METHODS

Chemicals. Glass wool, sodium citrate, dimethyl sulfoxide (DMSO), formic acid (FA), acetonitrile (ACN), the specific renin inhibitor Z-Arg-Arg-Pro-Phe-His-Sta-Ile-His-Lys-(Boc)-OMe, the ACE-I inhibitor captopril, ethanol, papain from Carica papaya (activity ≥3 U/mg), and all of the chemical reagents and stains used for microscopy analysis were obtained from Sigma-Aldrich (Dublin, Ireland). WPH BioZate 1 was supplied by Davisco Foods International (MN, USA). The renin inhibitor screening assay kit was supplied by Cambridge BioSciences (Cambridge, England, UK), and the ACE-I inhibition assay kit was supplied by NBS Biologicals Ltd. (Cambridgeshire, England, UK). For the breadmaking process, wheat flour (Shackleton’s Millers, Co. Meath, Ireland), salt, emulsified bread fat (Irish Bakels Ltd., Dublin, Ireland), and dried yeast (Doves Farm, UK) were used. Blood Collection and Fractionation Procedure. Whole bovine blood was collected at time of slaughter under hygienic conditions at the abattoir at the Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland. All animals slaughtered were female, Charolais cross heifer breeds and were aged between 23 and 24 months at the time of slaughter. Sodium citrate solution was used as an anticoagulant and was added immediately to blood following collection at a final concentration of 1.50% (w/v). Blood was chilled to 4 °C and handled carefully to minimize hemolysis. Plasma was separated from whole blood cells by centrifugation at 4 °C and 10,000g 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). Plasma proteins were precipitated from the freeze-dried extract using cold ethanol precipitation following a previously described method.29 Briefly, dehydrated plasma proteins were resuspended in Milli-Q water to a final concentration of 35 g/L. The pH was adjusted using 0.1 M HCl, and ethanol was added as the fractionation agent to separate plasma proteins. A fraction rich in fibrinogen (FI) was obtained by precipitation following addition of ethanol to plasma at a final concentration of 8% (v/v) and adjustment 1742

DOI: 10.1021/acs.jafc.5b06078 J. Agric. Food Chem. 2016, 64, 1741−1750

Article

Journal of Agricultural and Food Chemistry Table 1. Composition of Breads Formulated with Hydrolysates formulation

wheat flour (g)

water (g)

salt (g)

fat (g)

improver (g)

yeast (g)

hydrolysate (g)

control 4% WPH 4% GPH

150 144 144

93.7 94.2 95.1

3 3 3

1.5 1.5 1.5

1.5 1.5 1.5

2.25 2.25 2.25

0 6 6

glutaraldehyde in 80% ethanol. The fixative was replaced after 2 h with 80, 90, 95, and 100% ethanol in sequential steps for 1 h each. Fixed bread cubes were transferred to flat-ended Beem capsules containing LR White polymethacrylate resin (Agar Scientific Ltd., Stansted, UK) and allowed to infiltrate at room temperature overnight. The resin was replaced with fresh resin and capsules containing the fixed bread cubes were cured at 60 °C overnight in an oven. Semithin sections (5 μm thick) were then cut from the resin blocks on a Leica EM UC7 ultramicrotome (Leica Microsystems, Mannheim, Germany) and transferred to light microscope slides prior to imaging. Sections were cut from two replicate blocks. Bright Field Light Microscopy. Resin sections were double stained with one drop of an aqueous solution of 0.1% iodine in 0.2% (w/w) potassium iodide to stain starch for five seconds. The stain was then drained off, and one drop of 0.1% (w/w) acid fuschin (aq) was added. A coverslip was placed on top, and the sections were imaged using an Olympus BX51 light microscope (Mason Technology, Dublin, Ireland) using bright field illumination. Digital images (8 bit, TIFF, 2048 × 1024 pixel) were taken using a ProgRes CT3 camera (Jenoptik, Germany) with a 10× objective. Confocal Scanning Laser Microscopy. Resin sections of the fixed bread cubes were triple labeled to show the major ingredients. Briefly, one drop of 0.1% (w/w) ethanolic solution of fluorescein isothiocynate (FITC) was added to the resin section to label starch. After ten seconds, the FITC was drained off and replaced with one drop of fluorescent brightener 28 (FB28; 0.125% aq, w/w) to label cellulosic material, and then one drop of 0.1 (w/w) aq Fast Green FCF (FG) to label proteins. The sections were rinsed gently with running water, and a coverslip was placed on top. Stained sections were imaged using a Leica SP5 confocal scanning laser microscope (Leica Microsystems, Mannheim, Germany) fitted with a 63× 1.4 NA oil immersion objective. Sequential images were acquired using triplechannel imaging: a 405 nm blue diode laser to excite FB28, 488 nm argon laser excitation for FITC, and a 633 nm helium−neon laser for FG. Emission signals for FB28, FITC, and FG were sequentially collected using band-pass filters of 450−490, 510−550, and 650−700 nm, respectively. Digital 8-bit images (1024 × 1024 pixel) were obtained for each separate excitation wavelength, and channels were combined and pseudocolored to show cellulosic material (blue), starch (green), and protein (red/pink). Assessment of the Antihypertensive Effect of the GPH and the 4% GPH Bread in Vivo. All in vivo experiments were performed according to protocols approved by the University of Manitoba Animal Care Protocol and Management Review Committee in accordance with the Canadian Council on Animal Care Regulations. Male SHRs (20 weeks old) weighing between 290 and 300 g were kept at the Animal Care Facility of the Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba. SHRs were housed individually using steel cages in a room maintained under a 12 h day/ night cycle, temperature of 21 ± 2 °C, and relative humidity of 50%. The SHRs were divided into four groups of four rats each and were administered the following treatments: (a) GPH dissolved in phosphate buffer saline (PBS; pH 7.2) at 200 mg of protein/kg of body weight, (b) 4% GPH bread dissolved in PBS (pH 7.2) at 200 mg of protein/kg of body weight, (c) the positive control (captopril) dissolved in PBS at 10 mg/kg of body weight, and (d) PBS alone (negative control). Captopril is a common antihypertensive drug that has been used previously as a positive control for the assessment of hypotensive effects in SHRs by using a single oral treatment at a dose of 10 mg/kg of body weight.32 For the 4% GPH bread sample, the bread was dissolved in PBS and centrifuged, and the supernatant was gavaged. Each group received a 1 mL dose of each treatment via oral

FLUOstar Omega microplate reader (BMG LABTECH GmbH, Offenburg, Germany) at 450 nm. ACE-I IC50 values were determined for active hydrolysates by plotting the percentage of inhibition as a function of the concentration of test compound. Breadmaking. Bread loaves were produced following a straight dough baking procedure. Bread doughs were prepared for mixing according to the formulations listed in Table 1. Breads containing GPH or WPH were labeled as 4% GPH and 4% WPH, respectively. The amount of water added and the optimal mixing time were calculated using a Chopin MixOlab (Chopin Technologies, Villenueve-la-Garenne, France). After mixing, the doughs were placed in a Koma SDCC-1P/W proofer (Koma Koeltechnische Industrie B.V., Roermond, The Netherlands) at 30 °C and 80% relative humidity for 15 min. The bread pieces were then divided into 60 g pieces, molded by hand, placed in tins (9 × 6 × 4 cm), and proofed for a further 45 min period. The loaves were baked at 200 °C for 20 min. Four control and four hydrolysate-containing loaves were produced per batch. The loaves were allowed to cool for 2 h, placed in plastic bags, and stored at room temperature. The breads were analyzed at days one and six post-baking. Bread Analysis. Color recordings were taken on bread loaves using a Chroma Meter CR-400 (Konica Minolta, Tokyo, Japan). Crust and crumb CIE values were recorded in terms of L* (lightness), a* (redness/greenness), and b* (yellowness/blueness). Calibration was carried out with the white calibration plate CR-A40 provided by the manufacturer, and measurements were carried out using the D65 illuminant, which approximates to daylight. All samples were analyzed in triplicate on day one post-baking. Bread loaf volume was calculated using a BVM-L370 volume measurer (TexVol Instruments, Vike, Sweden). The results are the average of three samples and were measured at day one post-baking for all loaves. Image crumb analysis of bread slices was conducted by C-Cell (Calibre Control International Ltd., Warrington, UK). The instrument was connected to a PC running C-Cell software version 2.0. The samples were sliced by hand to 10 mm thickness and were measured at day one post-baking. Texture profile analysis was measured using a TA-XT2i Texture Analyzer (Stable Microsystems, Surrey, UK) equipped with a P/20P 20 mm Perspex cylinder probe. The test was performed at days one and six post-baking. Bread crumb moisture was calculated at days one and six after baking with a two-step AACC air-oven method 44-15. This method determines moisture content as loss in weight of a sample when heated under specified conditions. The moisture oven (Brabender Corp., Duisburg, Germany) was used to heat the milled crumb cut from a central plug from the center slices at 130 °C for 1 h. The water activity (aw) was measured in triplicate using an AquaLab LITE Water Activity Meter (Decagon Devices Inc., Pullman, WA, USA). The equipment was calibrated using 6.0 mol/kg of NaCl in H2O solution with a water activity value of 0.760 (Decagon Devices Inc., Pullman, WA, USA). Proteins from bread were extracted following the method of Galland-Irmouli et al.31 The total protein content of the hydrolysates and breads was determined in triplicate using a LECO FP628 Protein analyzer (LECO Corp., MI, USA) based on the Dumas method and according to AOAC method 992.15. Microscopy. For examination by bright field light microscopy and confocal scanning laser microscopy, bread samples were resin embedded, sectioned, and stained as described previously.24 Briefly, bread samples 3 mm3 in size were cut from a bread slice using a razor blade and placed in a fixative solution comprising 2.5% (w/w) 1743


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Journal of Agricultural and Food Chemistry Table 2. CIE Color Values of Crust and Crumba crust

a

sample

L*

control 4% WPH 4% GPH

56.6 ± 1.6 49.5 ± 1.2a 47.2 ± 1.2a

crumb

a* b

b*

16.5 ± 0.5 20.1 ± 0.3c 19.1 ± 0.0b a

L*

36.4 ± 0.1 33.0 ± 2.2a 34.1 ± 2.6a a

a*

74.2 ± 0.1 71.6 ± 2.5a 72.0 ± 2.4a a

b*

0.12 ± 0.11 0.05 ± 0.05a 0.03 ± 0.06a a

16.2 ± 0.3a 16.8 ± 0.5a 17.3 ± 0.6a

Values with different letters within each parameter have mean values that are significantly different (p < 0.05).

gavage. Before blood pressure was determined, rats were first restrained in a chamber at 40 °C with 4% isofluorane for 3 min to avoid stress-related blood pressure effects. The SBP of the SHRs was measured under normal atmospheric conditions at 0, 2, 4, 6, 8, and 24 h by tail cuff plethysmography (Mouse Rat Tail Cuff Blood Pressure System, IITC Life Sciences, Woodland Hills, CA, USA). The change in SBP was determined by subtracting the SBP at time n (where n was equal to 2, 4, 6, 8, and 24 h) from the baseline SBP (SBP at time 0). Statistical Analysis. Volume, aw, moisture, color, and texture analysis were replicated three times; mean values and standard deviations were calculated, and data were subjected to analysis of variance. For in vivo results, the repeated measures general linear model was used to test differences in SBP between treatments with the effects of time, treatment, and time treatment interaction included in the model, and posthoc Games−Howell tests were used to check the differences. The criterion for statistical significance was p < 0.05 in all cases. All statistical analyses were performed using SPSS for Windows v.18.0.

prediction of the degree of bioactivity but a prediction of how likely the peptides are to be bioactive. The in vitro ACE-I and renin inhibitory activities of the generated GPH were calculated. At a concentration of 1 mg/ mL, the GPH inhibited ACE-I by 69.1 ± 1.3% compared to the positive control, which had an ACE-I inhibitory activity of 88.1 ± 4.7% in vitro. The ACE-I IC50 value of the GPH was calculated and determined to be 0.95 ± 0.01 mg/mL. The ACE-I-inhibiting activity of the GPH generated herein compared favorably to that obtained previously from blood protein hydrolysates. Lafarga et al.34 recently reported a papain hydrolysate of a fibrinogen-rich fraction that inhibited ACE-I by half at a concentration of 1.86 mg/mL. Moreover, Deng et al.35 suggested pepsin as the most active enzyme for the generation of ACE-I-inhibiting hydrolysates of porcine hemoglobin, which had an ACE-I IC50 value of 1.53 ± 0.03 mg/mL. ACE-Iinhibiting hydrolysates were also generated previously from porcine red blood cells, whole plasma, and defibrinated plasma by hydrolysis in a continuous enzymatic membrane reactor using different enzymes.36 ACE-I IC50 values obtained in this study ranged from 0.58 to 4.10 mg/mL. In addition, Hyun and Shin37 generated enzymatic hydrolysates of whole bovine plasma, serum albumin, and globulins that showed ACE-Iinhibiting properties. However, the IC50 values of the Alcalase and trypsin hydrolysates of bovine globulins were calculated as 7.11 and 8.14 mg/mL, respectively. At a concentration of 1 mg/mL, the GPH inhibited renin by 45.6 ± 0.4%. The renin IC50 value was calculated to be 1.22 ± 0.22 mg/mL. The in vitro activity of the GPH generated herein was similar to those obtained for flaxseed protein hydrolysates where renin IC50 values ranging from 1.22 to 2.81 mg/mL were reported38 and to those obtained from Alcalase hydrolysates of kidney bean protein, which were found to inhibit renin by 20− 40% at a concentration of 1 mg/mL previously.39 However, the activity of the generated hydrolysate was lower compared to that obtained by Malomo et al.,40 who hydrolyzed hemp seed protein with the enzymes pepsin, Alcalase, papain, and pancreatin at different concentrations and obtained renin inhibitory values of over 60% for most of the studied hydrolysates. Inclusion of the Generated Hydrolysate into Bread: Physical Properties. Dough rheological properties and water absorption were calculated using a Chopin MixOlab. Values obtained for the 4% GPH breads showed no significant difference in dough development time and stability compared to the control bread following inclusion of GPH in flour at a concentration of 4%. A slight increase in the water absorption percentage was observed in the 4% GPH and the 4% WPH breads compared to the control. The water absorption was calculated as 62.5 ± 0.7, 62.9 ± 0.4, and 63.4 ± 0.1% for the control, 4% WPH, and 4% GPH breads, respectively. All of the breads were produced following a straight dough baking procedure. The result was a well-proportioned symmetrical loaf with a well-rounded top and an even, brown crust.

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RESULTS AND DISCUSSION Isolation of Bovine α- and β-Globulins: Characterization and in Vitro Bioactivity. The yield of FIII was calculated per liter of blood and was 10.2 ± 0.1 g/L. α- and βglobulins constitute approximately 0.51 and 0.53% of the total weight of bovine blood, respectively.12 The total protein content of the α- and β-globulin fraction was 88.8 ± 0.1%. Globulins comprise a heterogeneous group of proteins that include immunoglobulins, carrier proteins, and enzymes.33 In this study, 76 different proteins were identified in the isolated protein fractions. Identified proteins included prothrombin (P00735|THRB_BOVIN), beta-2-microglobulin (P01888| B2MG_BOVIN), beta-2-glycoprotein (P17690|APOH_BOVIN), and alpha-2-antiplasmin (P28800|A2AP_BOVIN). Bovine blood proteins, including fibrinogen, hemoglobin, and serum albumin, were previously used for the generation of bioactive peptides, but to date, to the best of our knowledge, no ACE-I or renin inhibitors have been generated from bovine globulins. Peptides within FIII were was identified by LC-MS/ MS. Identified peptides included HEHRFPLGPVT and TSPDRVFFR, which corresponded to f(77−87) and f(260−278) of alpha-1B-glycoprotein (Q2KJF1|A1BG_BOVIN) and the peptides FLQDMGLKAF and VYNLLPVK, which corresponded to f(671−680) and f(618−624) of alpha-2-macroglobulin (Q7SIH1|A2MG_BOVIN). Although the effect of each individual peptide on the overall observed bioactivity was not the aim of this study, the potential bioactivity of the identified peptides was predicted in silico using PeptideRanker.30 PeptideRanker is a server for the prediction of bioactive peptides based on a novel N-to-1 network, which is useful for identifying, among a set of peptides, those that are more likely to be bioactive. PeptideRanker scores were calculated for each peptide. Numerous peptides, including FSPFR, FNRPF, GRLPFFG, FNRPFL, DQIHFFF, and SGFSPFR showed scores ranging between 0.95 and 1.00. These scores are not a 1744


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properties would translate to similar mouth feel and texture when chewed by the consumer.24 Although no differences were observed in the texture profile of the breads, a significant decrease was observed in the moisture content between the 4% GPH bread and the control (p < 0.05). This resulted in a significantly lower decrease in moisture content in the 4% GPH bread compared to the control. No significant differences were observed in the moisture content of all breads at day six postbaking. Light micrographs of stained sections of the different bread formulations are shown in Figure 3. Starch was stained as a dark blue/black color, and protein appears in the figure as red/pink. The protein phase was found to be distributed in the bread matrix as a semicontinuous network and in a similar manner for all three samples. In addition, light micrographs of the 4% WPH and the 4% GPH bread samples showed similar starch granulation properties compared to those of the control. Confocal micrographs of resin embedded sections of the different bread formulations are shown in Figure 4. Cellulosic material, starch, and protein were imaged using FB, FITC, and FG, respectively, and are shown in the figure in blue, green, and pink/red, respectively. Results correlate well with those shown in Figure 3. The protein network was again found to be distributed as a semicontinuous network, and the cellulosic material was randomly scattered throughout the resin section. Overall, although the 4% GPH bread seemed to be denser, the matrix of the bread was generally similar in all three samples, and the addition of WPH and GPH to bread at a concentration of 4% did not alter the quality of the product. In addition, proteins from the 4% GPH and the control bread were extracted, and these were assessed for in vitro renin and ACE-I inhibitory activities. Results suggested that the bioactivity of the protein hydrolysate was not significantly altered by the breadmaking process as a significant increase of the renin and ACE-I inhibitory activity was observed in the 4% GPH bread compared to the control (p < 0.05). Proteins isolated from the GPH-containing bread inhibited renin and ACE-I by 12.2 ± 1.1 and 65.9 ± 1.4% compared to the proteins isolated from the control bread, which inhibited renin by 7.7 ± 0.9% and ACE-I by 56.4 ± 0.3%. Antihypertensive Activity in Vivo. Results shown in Figure 2 represent the short-term changes in SBP of SHRs observed over a 24 h period. SHRs were orally administered with the generated GPH, the 4% GPH bread, a positive control (captopril), and a negative control (saline solution). All of the animals were comparably hypertensive at time zero. The increase in SBP was significantly affected by time (p < 0.001), treatment (p < 0.001), and time*treatment (p < 0.001); thus, differences in the change in SBP were analyzed at each time point for the different treatments. Handling and multiple anesthetics during a 24 h period may cause stress-induced changes in blood pressure. However, this study uses anesthesia purely to restrain the rats. The procedure was optimized to ensure that the SHRs regained consciousness within 3−4 min of being taken out of the chamber, and the blood pressure of the animals was measured when the SHRs were no longer under isofluorane treatment and were under atmospheric conditions. This gave enough time for the measurement of blood pressure while the animals were still restrained and ensured neglible or no depression of SBP by the inhalational of the anesthetic agent. Results demonstrated that the SBP of the group of animals receiving the basal diet did not vary over a 24 h period. After 2

Volume and color are key quality parameters in bread products. No significant variations were observed in the loaf volume after the addition of WPH and GPH at a concentration of 4% compared to the control. Moreover, as shown in Table 3, no differences were observed in the specific volume after addition of GPH at a concentration of 4%. Surprisingly, the addition of WPH at a concentration of 4% resulted in an increase in the specific volume compared to the control (p < 0.05). These results contrast with previous studies where addition of protein hydrolysates resulted in a reduced loaf volume.24 Moreover, crust and crumb color of the three bread formulations were analyzed at day one post-baking, and L*, a*, and b* values were measured (Table 2). The parameter L* defines lightness, a* denotes the red/green value, and b* the yellow/blue value. An L* value between 0 and 50 indicates dark, and a value ranging between 51 and 100 indicates light. Moreover, positive a* and b* values indicate red and yellow, respectively, and negative a* and b* values indicate green and blue. 4% WPH and 4% GPH breads were found to have a crumb color presenting no significant variations compared to that of the control. However, significant differences were observed between the crust color in the control and in the 4% WPH and 4% GPH breads. These breads were significantly darker than the control (p < 0.05), and presented L* values of 47.2 ± 1.2 and 49.4 ± 1.2, respectively, compared to the control (56.64 ± 1.60). The addition of protein hydrolysates to bread formulations may result in an increase of Maillard reactions in the crust and, in turn, in a darker crust color.24 Structural properties of the crumb are of key importance in terms of the sensory properties of bread and directly affect staling and texture properties. Results of the image crumb analysis are presented in Table 3. The addition of WPH and Table 3. Image Crumb Analysisa sample control 4% WPH 4% GPH

number of cells

cell diameter (mm)

average cell elongation

wall thickness (mm)

1585 ± 63a 1572 ± 24a

2.20 ± 0.28a 2.15 ± 0.05a

1.47 ± 0.01a 1.45 ± 0.02a

0.44 ± 0.01a 0.45 ± 0.00a

1566 ± 72a

2.19 ± 0.13a

1.45 ± 0.00a

0.45 ± 0.01a

a

Values with different letters within each parameter have mean values that are significantly different (p < 0.05).

GPH had no significant effect on the number of cells, area of cells, and wall thickness of the breads. In addition, the results of the texture profile analysis of bread crumb, water activity, and moisture content are shown in Figure 2. Water activity is an important measure of a products’ shelf life.41 No differences were observed between aw values in the control bread and the 4% WPH and 4% GPH breads at day one or day six postbaking. The expected decrease in aw at day six can be attributed to a decrease in moisture content due to bread staling. Moreover, no significant differences were observed at day one post-baking in the hardness, adhesiveness, springiness, chewiness, and cohesiveness of the 4% WPH and 4% GPH breads compared to the control (Figure 1). The effect of storage time is also represented in Figure 2. As expected, crumb hardness increased for all three formulations. The most significant increase in hardness between days one and six post-baking was observed in the 4% GPH bread. Crumb cohesiveness, springiness, and adhesiveness were also significantly affected due to bread staling. Comparable cohesiveness and springiness 1745


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Figure 1. Texture profile analysis, moisture content, and water activity at days one and six post-baking. Hardness, springiness, chewiness, cohesiveness, moisture content, and aw were measured at days one and six after baking. The values represent the means of three independent experiments ± standard error of the mean (SEM). Bars with different letters within days one and six post-baking indicate significant differences (p < 0.05).

h, a drop in SBP was observed in the rat groups that were administered the GPH, the 4% GPH, and the positive control when compared to the group receiving the basal diet. This

trend was also observed after 4, 6, and 8 h. After 24 h, the animals administered with the GPH showed an increased SBP, and the difference with the control was no longer appreciated. 1746


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Figure 2. Short-term antihypertensive effect on SHRs. Change in SBP of SHRs measured after oral administration of the GPH (200 mg of protein/kg of body weight) and the 4% GPH bread (200 mg of protein/kg of body weight) compared to the positive control (captopril, 10 mg/kg of body weight) and a negative control (saline solution) at different times. Results are expressed as means ± SEM. Different letters indicate statistically significant differences (p < 0.05) in the change in SBP between treatments measured at every time point (2, 4, 6, 8, and 24 h).

The animals receiving captopril and bread did not show statistically significant differences between them at any of the analyzed times in this study and were also similar to the animals receiving GPH until 24 h. Although captopril was orally administered at a dose 20 times less concentrated than the GPH or 4% GPH bread, the GPH, 4% GPH bread, and captopril showed a similar trend in lowering the SBP of the SHRs, suggesting the potential of the generated hydrolysate to lower SBP on a short term basis. Animals that were administered with captopril and 4% GPH bread did not show statistically significant differences between them at any of the times analyzed and were similar to the animals orally administered GPH until 24 h. The maximum decrease in SBP in rats administered with the GPH and the 4% GPH bread was 34.5 ± 3.4 and 36.3 ± 1.2 mmHg, respectively, and was observed 8 h after oral administration for both samples. Results obtained herein are consistent with previous studies where the hypotensive effect of captopril, even at lower doses, was significantly better after 24 h of oral administration.32 Similar results were obtained previously in SHRs with an enzymatic hydrolysate of hemp seed protein, where decreases of 20 and 30 mmHg in SBP were observed 2 and 8 h after oral administration at a dosage of 200 mg/kg of body weight.42 The drop in SBP observed in this study was lower than that obtained previously by Fitzgerald et al.43 who observed a drop of 34 mmHg in SBP 24 h after oral administration at a dose of 50 mg/kg of body weight. However, the GPH generated herein showed a higher hypotensive effect than a thermolysin

Figure 3. Light micrograph images of the control (A), 4% WPH (B), and 4% GPH (C) breads. Resin sections were double stained with iodide and acid fuchsin to stain starch (dark blue/black) and protein (pink).

hydrolysate of pea protein, where a drop of 19 and 13 mmHg in SBP after 4 and 8 h of oral administration (200 mg/ kg of body weight) was observed.44 Rising demand for protein worldwide gives the Irish beef industry an opportunity to expand its export markets. The recovery of blood proteins and their subsequent processing into high-value bioactive hydrolysates and peptides represents an 1747


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Figure 4. Confocal micrographs of the control (A), 4% WPH (B), and 4% GPH (C). Resin sections of the fixed bread cubes were triple labeled to show cellulosic material (blue), starch (green), and protein (red/pink).

economic opportunity for meat processors. In this study, bovine blood globulins were identified as a resource for the generation of bioactive peptide hydrolysates with observed antihypertensive activities in vivo in the SHRs model. The in

vitro and in vivo results obtained in this work suggest that a papain hydrolysate of bovine α- and β-globulins can rapidly reduce SBP and thus has potential for use as an ingredient in the functional foods market. Bovine blood is regularly 1748


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for the management of hypertension. Guidelines sub-committee of the World Health Organization. Clin. Exp. Hypertens. 1998, 21, 1009− 1060. (6) Guidelines Committee. 2003. European Society of Hypertension−European Society of Cardiology guidelines for the management of arterial hypertension*. J. Hypertens. 2003, 21, 1011−1053. (7) 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. (8) Kim, S.-K.; Wijesekara, I. Development and biological activities of marine-derived bioactive peptides: A review. J. Funct. Foods 2010, 2, 1−9. (9) Roy, F.; Boye, J. I.; Simpson, B. K. Bioactive proteins and peptides in pulse crops: Pea, chickpea and lentil. Food Res. Int. 2010, 43, 432−442. (10) Korhonen, H. Milk-derived bioactive peptides: From science to applications. J. Funct. Foods 2009, 1, 177−187. (11) Nagpal, R.; Behare, P.; Rana, R.; Kumar, A.; Kumar, M.; Arora, S.; Morotta, F.; Jain, S.; Yadav, H. Bioactive peptides derived from milk proteins and their health beneficial potentials: an update. Food Funct. 2011, 2, 18−27. (12) Bah, C. S. F.; 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) 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. (14) Hirata, H.; Nakamura, Y.; Yada, H.; Moriguchi, S.; Kajimoto, O.; Takahashi, T. Clinical effects of new sour milk drink on mild or moderate hypertensive subjects. J. New Rem & Clin 2002, 51, 61−69. (15) Mizuno, S.; Matsuura, K.; Gotou, T.; Nishimura, S.; Kajimoto, O.; Yabune, M.; Kajimoto, Y.; Yamamoto, N. Antihypertensive effect of casein hydrolysate in a placebo-controlled study in subjects with high-normal blood pressure and mild hypertension. Br. J. Nutr. 2005, 94, 84−91. (16) Nakamura, Y.; Kajimoto, O.; Kaneko, K.; Aihara, K.; Mizutani, J.; Ikeda, N.; Nishimura, A.; Kajimoto, Y. Effects of the liquid yogurts containing “lactotripeptide (VPP, IPP)” on high-normal blood pressure. J. Nutr. Food 2004, 7, 123−137. (17) Seppo, L.; Jauhiainen, T.; Poussa, T.; Korpela, R. A fermented milk high in bioactive peptides has a blood pressure-lowering effect in hypertensive subjects. Am. J. Clin. Nutr. 2003, 77, 326−330. (18) Kawasaki, T.; Jun, C. J.; Fukushima, Y.; Kegai, K.; Seki, E.; Osajima, K.; Itoh, K.; Matsui, T.; Matsumoto, K. Antihypertensive effect and safety evaluation of vegetable drink with peptides derived from sardine protein hydrolysates on mild hypertensive, high-normal and normal blood pressure subjects. Fukuoka Igaku Zasshi 2002, 93, 208−218. (19) EFSA. Scientific opinion on the substantiation of a health claim related to isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP) and maintenance of normal blood pressure pursuant to Article 13(5) of Regulation (EC) No 1924/2006. EFSA Journal 2011, 9, 18. (20) Ramalho Ribeiro, A.; Gonçalves, A.; Colen, R.; Nunes, M. L.; Dinis, M. T.; Dias, J. Dietary macroalgae is a natural and effective tool to fortify gilthead seabream fillets with iodine: Effects on growth, sensory quality and nutritional value. Aquaculture 2015, 437, 51−59. (21) Lafarga, T.; Gallagher, E.; Walsh, D.; Valverde, J.; Hayes, M. Chitosan-containing bread made using marine shellfishery byproducts: Functional, bioactive, and quality assessment of the end product. J. Agric. Food Chem. 2013, 61, 8790−8796. (22) Yep, Y. L.; Li, D.; Mann, N. J.; Bode, O.; Sinclair, A. J. Bread enriched with microencapsulated tuna oil increases plasma docosahexaenoic acid and total omega-3 fatty acids in humans. Asia Pac. J. Clin. Nutr. 2002, 11, 285−291. (23) Crider, K. S.; Bailey, L. B.; Berry, R. J. Folic Acid Food FortificationIts History, Effect, Concerns, and Future Directions. Nutrients 2011, 3, 370−384. (24) Fitzgerald, C.; Gallagher, E.; Doran, L.; Auty, M.; Prieto, J.; Hayes, M. Increasing the health benefits of bread: Assessment of the

consumed in numerous countries and blood proteins have already been used for supplementation of baked products.45 Moreover, the in vivo hypotensive effect of the 4% GPH bread, and the in vitro ACE-I inhibitory activity of the proteins isolated from this bread, demonstrated that the antihypertensive activity of GPH was resistant to the breadmaking process, which includes baking and high temperatures, and shows potential for use in other food applications. This study also highlights that baked products may be suitable vehicles for delivery of bioactive compounds and demonstrates that the hypotensive activity of the papain hydrolysate of bovine globulins was resistant to the high temperatures found during the baking process. In addition, numerous peptides were identified in the GPH generated in this study. However, the complete composition of the GPH was not properly reflected, and the effect of each individual amino acid on the observed antihypertensive activity was not calculated.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +353 (0) 1 8059957. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.L. is a Teagasc Walsh Fellowship recipient. This work forms part of the ReValueProtein Research Project (Grant Award No. 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 the National Development Plan 20072013. The authors thank Dr. Adeola Alashi, Dr. Sunday Malomo, and Ifeanyi Nwachukwu for technical assistance during the in vivo experiments.

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ABBREVIATIONS USED WHO, World Health Organization; SBP, systolic blood pressure; DBP, diastolic blood pressure; ESH, European Society of Hypertension; ACE-I, angiotensin-I-converting enzyme; RAAS, renin-angiotensin-aldosterone system; EFSA, European Food Safety Authority; GPH, globulin protein hydrolysate; WPH, whey protein hydrolysate; SHRs, spontaneously hypertensive rats; DMSO, dimethyl sulfoxide; FA, formic acid; ACN, acetonitrile; SD, standard deviation; aW, water activity; FITC, fluorescein isothiocynate; FB28, fluorescent brightener 28; FG, Fast Green; PBS, phosphate buffer saline; SEM, standard error of the mean

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Addition of an Enzymatic Hydrolysate of Bovine Globulins to Bread

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