Inhibitory Properties of Kidney Bean Protein Hydrolysate and its Membrane Fractions Against Renin, Angiotensin Converting Enzyme, and Free Radicals

Abstract

Kidney bean hydrolysate (KBH) was obtained by alcalase hydrolysis of the seed globulin protein followed by membrane ultrafiltration to produce peptide fractions that differ in molecular sizes (<1, 1–3, 3–5, and 5–10 kDa). Evaluation of potential antihypertensive properties of the peptides showed that the <1 and 5–10 kDa fractions exhibited significantly highest (p<0.05) renin inhibition. In contrast, the KBH and peptide fractions showed similar and non–significant (p<0.05) inhibitory activities against angiotensin converting enzyme. The antioxidant power of the hydrolysates was evaluated through free radical scavenging activities (DPPH and hydroxyl radical), inhibition of iron activities (metal chelation and ferric reducing antioxidant power) and inhibition of linoleic acid peroxidation. The <1 and 5–10 kDa peptide fractions showed significantly (p<0.05) higher ability to scavenge DPPH free radical, inhibit peroxidation oflinoleic acid and reduce Fe³⁺ to Fe²⁺. Generally the fractions with <1 and 5–10 kDa peptides showed better potential as antihypertensive and antioxidant peptides, probably due to their slightly higher contents of hydrophobic aminoacids. It was concluded that kidney bean protein hydrolysate and some of the peptide fractions could potentially serve as useful ingredients to formulate functional foods and nutraceuticals against hypertension and oxidative stress.

Keywords: Kidney bean; protein hydrolysate; alcalase; renin; membrane ultrafiltration; angiotensin converting enzyme; antioxidant activity

Introduction

Several food proteins and peptides have been shown to display specific biological activities in addition to their proven nutritional value [1–3]. A growing body of scientific evidence reveals the positive impact of bioactive peptides and proteins on body function and human health by alleviating conditions such as coronary (ischemic) heart disease, stroke, hypertension, cancer, obesity, diabetes, and osteoporosis [4,5]. Specifically, research studies have shown some evidence for the efficiency of plant protein–derived peptides in improving hypertension or contributing to the overall antioxidant capacity of cells [6–9]. It has been reported that a large range of antihypertensive and antioxidant peptides and peptide mixtures (hydrolysates) have been produced from various food products such as beans, soy, corn, potato, peanut, milk, whey, egg, and meat proteins [3]. These peptides are inactive within the sequence of their parentproteins but can be released by chemical, enzymatic and microbial methods [3,10]. By far, the most effective and dependable method to produce peptides with the intended functionalities is enzymatic digestion [3]. Bioactive peptides are either produced in vivo by the action of gastrointestinal enzymes or obtained in vitro using specific enzymes, or during the preparation of certain foods. However, the source of proteins, the protein substrate pretreatment, the type ofenzymes used, and the hydrolysis conditions applied, all affect the efficacy of protein hydrolysates and type of peptides produced [3]. It is also known that the nature of residues in a peptide influences itsactivity. These peptides have the advantage of being naturally derived from food protein sources normally consumed as part of the daily diet, and they are considered to be milder and safer without the side effects associated with drugs. Peptides with antioxidant and ACE–inhibitory activities are usually rich in hydrophobic amino acids, which enhance absorption and interaction with target enzymes or free radicals [3,11].

High blood pressure confers a high risk of complications, as it is one of the major risk factors for cardiovascular diseases including coronary heart disease, peripheral artery disease and stroke [11,12]. Clinical evidence has shown that peptides released by the action of enzymes could be involved in the inhibition of the reninangiotensin– aldosterone system (RAAS), which is one key pathway for combating hypertension. In the RAAS, kidney–secreted renin cleaves angiotensinogen to produce an inactive decapeptide called angiotensin I; angiotensin converting enzyme (ACE) then removes a dipeptide from the C–terminal of angiotensin I to generate angiotensin II, a very potent vasoconstrictor that also enhances sodium (fluid) retention [11]. In addition, ACE is also responsible for inactivating the vasodilator bradykinin [13]. For this dual role in the maintenance of blood pressure and fluid and electrolyte homeostasis, inhibition of ACE has been successfully used for the treatment of hypertension and congestive heart failure. Synthetic ACE inhibitors such as captopril, enalapril, lisinopril and ramipril or the renin inhibitor (aliskiren) have been widely used for the effective clinical treatment of hypertension and heart failure in humans. However uses of these drugs are also associated with disadvantages, such as diarrhea, coughing, allergies,taste disturbances, skin rashes, impaired renal function, and in some cases excessively low blood pressure, i.e. hypotension [14–17]. For this reason, identification of possible natural sources of ACE inhibitors that have a strong antihypertensive activity and resistance to digestion by various proteases and with minimal negative side effect will be of great interest to formulators of functional foods. Although the effectiveness of the ACE–inhibitory activity may not be as high as those of synthetic drugs, many natural ACE–inhibitory peptides isolated from different food proteins could be applied in the prevention of hypertension and in the initial treatment of mildly hypertensive individuals [18].

Peptides have also been shown to be capable of inhibiting the uncontrolled oxidation of the biomacromolecules usually caused by reactive oxygen species (ROS). Peptides are known to act against an oxidative sequence by terminating chain reactions and removing free radical intermediates; therefore, they are able to reduce intensity of oxidative stress–related diseases like cancer, heart disease etc. Bioactive peptides block the oxidation process by neutralizing free radicals such as superoxide anion radical (O₂⁻) and hydroxyl radical (.OH) which are products of regular metabolism [19]. Several in vitro studies have produced evidence that peptides generated from certain food proteins by enzymatic hydrolysis, including quinoa seed proteins [20], capelin protein [21], canola [22] and egg white protein [23] possess strong antioxidant activities. In particular, published studies have revealed strong evidence for the antioxidant activity for legume seed protein–derived peptides such as those from chickpeas [18,24– 26], cowpea [27], and soybean [28], but there is scanty information on the in vitro antioxidant and antihypertensive activity of kidney bean protein hydrolysate and the effect of peptide size on potency.

Kidney bean (Phaseolus vulgaris) is a pulse crop that contains high amount of proteins (20-30%) on a dry weight basis [29]. This puts them among some of the richest food sources of proteins, making them a good candidate to explore for the production of bioactive peptides. The goal of this study was to obtain an enzymatic protein hydrolysate from kidney bean globulin (major seed protein fraction) and fractionate the inherent peptides according to size using a membrane ultrafiltration system. The protein hydrolysate and membrane ultrafiltration fractions were then analyzed for in vitro antihypertensive (inhibition of ACE and renin) antioxidant (free radical scavenging, inhibition of linoleic acid oxidation, iron chelation, and ferric iron reducing power) activities.

Materials and Methods

Materials

Red kidney bean seeds were obtained from a local store in Winnipeg. Alcalase, N–(3–[2–furyl]acryloyl)–phenylalanylglycylglycine (FAPGG), glutathione (GSH), DPPH (2, 2–Diphenyl–1– picrylhydrazyl), 1–anilino–8–naphthalene sulfonate (ANS) and ACE (from rabbit lung) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Human recombinant Renin Inhibitor Screening Assay Kit was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Other analytical grade reagents and ultrafiltration membranes were obtained from Fisher Scientific (Oakville, ON, Canada).

Extraction and isolation of globulin protein

Red kidney bean seeds were ground into flour using a Retsch ZM200 centrifugal mill (Retsch GmbH, Haan, Germany). Globulinproteins were extracted from the flour according to the previously described ammonium sulfate precipitation method [30]. Briefly, an aqueous extract (obtained using 0.1 M phosphate buffer, pH 7.0 containing 0.4 M NaCl) of the flour was adjusted to 40% ammonium sulfate saturation, in order to precipitate smaller proteins and enzymes. After centrifugation (7000xg, 1 h, 4°C), the supernatant was then brought to 80% ammonium sulfate saturation to precipitate the globulins. The precipitating salt (ammonium sulfate) was then removed from the isolated globulins by dialyzing sample against water. The dialysis bag content was centrifuged (7000xg, 1 h, 4°C) and the resultant precipitate was freeze–dried as the globulin isolate.

Preparation and fractionation of kidney bean globulin protein hydrolysates

Proteolysis of the isolated kidney bean globulin isolate was conducted with alcalase. The globulin protein isolate (5%, w/v, protein weight basis) was suspended in deionized water in a reaction vessel equipped with a stirrer, heated to 50°C and adjusted to pH 9.0 prior to the addition of alcalase (4% w/w, based on the protein content of the protein isolate). The digestion was performed at the above stated conditions for 4 h with the pH of the reaction mixture maintained constant by addition of 2 M NaOH. At the end of the proteolysis period, the mixture was heated in boiling water for 10 min to inactivate alcalase; after cooling to room temperature, the mixture was adjusted to pH 4.0 with 2 M HCl to precipitate undigested proteins. Thereafter, the hydrolysate was centrifuged (30 min at 7,000xg). The supernatant containing target peptides was collected as the kidney bean protein hydrolysate (KBH) and a portion saved and stored at –20°C. The remaining liquid hydrolysate was passed through a 1 kDa membrane and the retentate passed through a 3 kDa ultrafiltration membrane. The retentate from 3 kDa membrane was passed through a 5 kDa whose retentate was then passed through a 10 kDa membrane. Permeates collected from each membrane were designated as <1, 1–3, 3–5 and 5–10 kDa peptide fractions, respectively. The KBH and membrane fractions were then freeze–dried and their protein contents determined by the modified Lowry method [31].

Amino acid analysis

An HPLC system was used for the analysis of the amino acid profiles after samples were hydrolyzed with 6 M HCl according to the method of Bidlingmeyer et al. [32]. The cysteine and methionine contents were determined after performic acid oxidation [33] while tryptophan content was determined after alkaline hydrolysis [34].

Surface hydrophobicity (So) determination

So of the KBH and the ultrafiltration peptide fractions was determined using a hydrophobic fluorescence probe, 1–anilino–8– naphthalene sulfonate (ANS) method as described by Hayakawa and Nakai [35] with some modifications. Samples were serially diluted to 0.0025–0.015% (w⁄v) in 0.01 M phosphate buffer (pH 7.0). Twenty µl of ANS (8.0 mM in 0.1 M phosphate buffer, pH 7.0) was added to 2 ml of sample solution. Fluorescence intensity of ANS–peptide conjugates was measured with an FP–6300 spectrofluorimeter (JASCO, Tokyo, Japan) at the excitation and emission wavelengths of 390 and 470 nm, respectively.

Determination renin–inhibitory activity

The method of Li and Aluko [6] was used to perform the renin inhibition assay using the Renin Inhibitor Screening Assay Kit. The background wells contained 20 µl of substrate, 160 µl of assay buffer, and 10 µl of Milli–Q. Thereafter, an aliquot of 20 µl of substrate, 150 µl of assay buffer, and 10 µl of Milli–Q water were added to the blank wells while 20 µl of substrate, 150 µl of assay buffer, and 10 µl of KBH or peptide fraction (final assay concentration of 1 mg protein⁄ml) were added to the inhibitor wells. The reaction was initiated by adding 10 µl of renin to the control and sample wells. The microplate was shaken for 10 s to mix, incubated at 37°C for 15 min, and then fluorescence intensity (FI) was recorded using an excitation wavelength of 340 nm and emission wavelength of 490 nm.

The percentage inhibition was calculated as: % Renin inhibition = FI (blank) – FI (sample) x 100⁄FI (blank)

Determination of ACE–inhibitory activity

The ACE–inhibitory activity was assayed as described by Udenigwe et al. [36] using FAPGG as substrate. Briefly, 1 ml of 0.5 mM FAPGG (dissolved in 50 mM Tris-HCl buffer containing 0.3 mM NaCl, pH 7.5) was mixed with 20 µl of ACE (1 U⁄ml; final activity of 20 mU) and 200 µl of KBH or peptide fractions (final assay concentrationof 1 mg protein⁄ml) in 50 mM Tris–HCl buffer. The decrease in absorbance at 340 nm, which is due to cleavage of the Phe–Gly peptide bond of FAPGG was recorded for 2 min at room temperature. For the blank experiment, Tris–HCl buffer was used instead of peptide fraction solutions. All experiments were performed in triplicate. Thepercentage inhibition of ACE was calculated as:

% ACE inhibition = Abs of blank–Abs of sample x 100⁄Abs of blank

DPPH radical scavenging assay

Reduction of DPPH by an antioxidant usually results in a loss of absorbance at 517 nm. The extent of discoloration of the solution indicates the scavenging efficiency of the added compound. Determination of antioxidant activity of KBH and the peptide fractions was adapted from the method described by Hou et al. [37] using a 96–well microplate. The KBH and the peptide fractions (final assay concentration of 1 mg protein⁄ml) were dissolved in 0.1 M sodium phosphate buffer, pH 7.0 containing 1% (w⁄v) Triton X–100. A solution of DPPH was prepared in methanol to a final concentration of 100 µM. Samples (100 µl) were added to 100 µl of DPPH in a 96– well microplate. A blank well contained only DPPH and the sodium phosphate buffer. The plate was then covered and incubated in the dark at room temperature for 30 min; absorbance of the sample (As)and blank (Ab) at 517 nm was measured in a spectrophotometer. The scavenging activities of KBH and the peptide fractions were compared to that of GSH. The percent scavenging activity of GSH and the samples was calculated using the following equation: DPPH Radical Scavenging Activity (%) = (Ab–As⁄Ab) x 100

Hydroxyl radical scavenging assay

The hydroxyl radical scavenging activity was measured according to the protocol previously described [38]. KBH, peptide fractions, GSH and 1, 10–phenanthroline (3 mM) were each separately dissolved in 0.1 M sodium phosphate buffer (pH 7.4) while FeSO₄ (3 mM) and0.01% hydrogen peroxide were each separately dissolved in distilled water. An aliquot (50 µl) of 1, 10–phenanthroline and 50 µl of FeSO4 were added consecutively to 50 µl of KBH, peptide fractions, GSH, or buffer (control) in a clear, flat bottom 96–well microplate. Final assay concentration of samples was 1 mg protein⁄ml. To initiate reaction in the wells, 50 µl of hydrogen peroxide (H₂O₂) solution was added to the mixture, which was then covered and incubated at 37°C for 1 h with shaking. Thereafter, the absorbance of the mixtures was measured at 536 nm every 10 min for a period of 1 h. The absorbance was also determined for a blank (without peptides and H2O2) and a blank(without peptides). The ?OH scavenging activity was calculated as described by Ajibola et al. [38].

Determination of Fe²⁺ chelating activity

The iron chelating activity of KBH and peptide fractions was measured following the ferrozine method as described by Ajibola et al. [38]. KBH, peptide fraction or GSH solutions (final concentration of 1 mg protein⁄ml) was mixed with 0.05 ml of 2 mM FeCl₂ and 1.85 ml distilled water in a reaction tube. Thereafter, 0.1 ml of 5 mM Ferrozine solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 10 min followed by removal of 200 µl aliquot of the reaction mixture and added to a clear bottom 96–well plate. The control experiment contained all the reaction mixtures except that distilled water was used to replace the sample. Absorbance of sample (As) and blank (Ab) was measured using a spectrophotometer at 562 nm and the metal chelating activity of the sample was compared to that of GSH. The percentage chelating effect (%) was calculated using the following equation:

Fe²⁺ chelating activity (%) = (Ab–As⁄Ab) x 100

Ferric reducing antioxidant power (FRAP) assay

The ability of the hydrolysate to reduce iron (III) was determined according to the method of Yildirim et al. [39] with some modifications. Different concentrations of KBH, peptide fractions or GSH (1, 5 and 10 mg⁄ml) in 250 µl of distilled water were mixed with phosphate buffer (250 µl of 0.2 mM, pH 6.6) and 250 µl of 1% potassium ferricyanide solution dissolved in distilled water. The mixture was incubated at 50oC for 30 min, followed by addition of 250 µl of 10% (w⁄v) trichloroacetic acid. The mixture was then centrifuged at 1000xg for 10 min. Finally, 250 µl of the supernatant solution was mixed with 50 µl of distilled water and 50 µl of 0.1% (w⁄v) ferric chloride solution followed by addition of distilled water (200 µl). After 10 min reaction the absorbance of the resulting solution was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.

Inhibition of linoleic acid oxidation

Linoleic acid oxidation was measured using a previously described method [38]. KBH, peptide fractions or GSH were dissolved in 1.5 ml of 0.1 M phosphate buffer, pH 7.0 at a final concentration of 1 mg protein⁄ml. Each mixture was added to 1 ml of 50 mM ethanolic linoleic acid and stored in a glass test tube kept at 60°C in the dark for 7 days. On a daily basis, 100 µl of the reaction mixture was removed and mixed with 4.7 ml of 75% aqueous ethanol, 0.1 ml of ammonium thiocyanate (30%, w⁄v) and 0.1 ml of 0.02 M acidified ferrous chloride (dissolved in 1 M HCl). An aliquot (200 µl) of the resulting solution was added to a clear bottom 96–well microplate and the degree ofcolor development was measured using the spectrophotometer at 500 nm after 3 min incubation at room temperature.

Statistical Analysis

Data were collected in triplicates and subjected to one way analysis of variance using Statistical Analysis System Software (SAS version 9.2, SAS Institute, Cary, NC). Significant differences were determined by Duncan’s multiple range test and accepted at p<0.05.

Result and discussion

Protein hydrolysis

Enzymatic proteolysis of kidney bean globular protein and subsequent fractionation of the resultant KBH by membrane ultrafiltration resulted in fractions rich in small size (<10 kDa) peptides. The percent gross yield of KBH was 78%, and approximately 30.7, 20.3, 17 and 18% of peptides in the KBH had molecular weights of <1, 1–3, 3–5 and 5–10 kDa, respectively. The final retentate (>10 kDa fraction), which contained large size peptides had a yield of 14%. The protein contents were ˜87, 96, 89, 78 and 90% for the <1, 1–3, 3–5, 5–10 kDa and the KBH, respectively. The high yield of KBH reflects defficient digestion of the globular proteins by alcalase.

Amino acid analysis

Amino acid analysis of the unhydrolyzed and alcalase–treated globulins from kidney bean seed as well as the peptide fractions collected as permeates from 1, 3, 5 and 10 kDa membrane cut–offs are shown in Table 1. The amino acid analysis of the unhydrolyzed globulin meets the FAO’s 35% recommendation for essential amino acid content [40]. Protease hydrolysis of the globulin proteins affected the amino acid content of the fractions in various ways. Generally, theunhydrolyzed globulin and KBH as well as the peptide fractions all contained low levels of methionine and cysteine, which is typical of legume proteins. Conversely, all samples had high contents of glutamic acid, glutamine, aspartic acid, asparagine, lysine and alanine. Except for proline, cysteine, isoleucine valine and histidine which were relatively higher, most of the amino acid contents of the KBH were slightly lower than the unhydrolyzed globulin. In contrast, Pownall et al. [41] investigated the amino acid composition of pea peptides fractions separated from the <3 kDa permeate using high performance liquid chromatography (HPLC) and found an increase in hydrophobic (both aliphatic and aromatic) amino acid of the hydrolysate when compared with the isolate. In our study, when compared to the KBH, most peptide fractions showed higher average surface hydrophobicity. Pownall et al. [41] and Megías et al. [24] also reported higher contents of certain hydrophobic amino acids in relation to the original proteinhydrolysate. Hydrolysis markedly increased the cysteine content of the KBH and all the peptide fractions even though the other sulphur–containing amino acid, methionine was slightly reduced in all the fractions. The total hydrophobic aliphatic amino acid (valine, isoleucine and leucine) content showed the highest concentration in the <1 kDa fraction. The percentage of valine also increased in the hydrolysate and in the polypeptides with 5–10 kDa size, but was least in the 3–5 kDa fraction. The percentage content of leucine and isoleucine residues as well as hydrophobic aromatic amino acid (phenylanaline and tyrosine) were highest in the <1 kDa fraction, decreasing as the membrane MW cut–off increased from 1 kDa to 10 kDa. Proline, aslightly hydrophobic amino acid increased in the peptide fractions as MW cut–off was increased. Asparagine⁄aspartic acid and glutamine⁄ glutamic acid contents were also increased in the peptide fractions with exception of the 5–10 kDa fractions.

Surface hydrophobicity

Proteolysis of proteins may cause changes in protein globular structure as the hydrophobic regions hidden within the native protein are exposed [42]. After proteolysis, more hydrophobic amino acids are exposed because of the catalytic specificity of alcalase, which cleaves peptide bonds formed by these specific amino acid residues [43]. Quantitative structure–activity relationship studies of ACEinhibitory peptides have shown that peptides composed of amino acids with strongly hydrophobic (or aromatic) side chains have potent ACE–inhibitory activities [44,45]. Hydrophobicity is also a very important contributing factor to the activity of antioxidative peptides. This is because, the surface hydrophobic site is partly responsible for formation and maintenance of the spatial structures as well as protein interactions, including binding to cell membranes, protein-protein recognition, and formation of complexes with biologically active compounds [46]. Since the hydrophobic interactions are the driving forces for manifestation of the physiological functions of peptides, information on the hydrophobic character of bioactive peptides could contribute to further understanding of their mechanism of action. To determine hydrophobicity of the peptides, a hydrophobic fluorescent dye (ANS) was used as a probe. As shown in Figure 1, the affinity of ANS for hydrophobic patches increased from the <1 kDa fraction to the 5–10 kDa. This is probably because there are more oligopeptides available as the size of the membranes increased with larger surface containing the exposed hydrophobic residues. Wu et al. [47] also observed a decrease in the surface hydrophobicity of smaller soypeptides prepared by longer time papain hydrolysis compared to the larger peptides produced by shorter time hydrolysis. The authors attributed the observed difference to fewer hydrophobic binding sites on the smaller peptides compared to the larger peptides. The same authors also observed lower surface hydrophobicity for the ultrafiltrates than the hydrolysates [47]. On a similar note Molina Ortiz et al. [48] also observed that smaller chain peptides had less surface hydrophobicity. However, our results differ from those reported by Wang and co–workers [49] for papain hydrolysates of wheat gluten who showed that the permeate with a molecular weight cut–off of 5 kDa had higher surface hydrophobicity than the hydrolysate. The trend for the surface hydrophobicity is not consistent with the hydrophobic amino acid residue content, which suggests that arrangement of the amino acids differ between the peptide chains.