JOURNAL OF MEDICINAL FOOD J Med Food 00 (0) 2015, 1–8 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2015.0007
FULL COMMUNICATION
ACE-I Inhibitory Activity from Phaseolus lunatus and Phaseolus vulgaris Peptide Fractions Obtained by Ultrafiltration David Betancur-Ancona,1 Gloria Da´vila-Ortiz,2 Luis Antonio Chel-Guerrero,1 and Juan Gabriel Torruco-Uco 2,3 1
Facultad de Ingenierı´a Quı´mica, Universidad Auto´noma de Yucata´n, Me´rida, Me´xico. Escuela Nacional de Ciencias Biolo´gicas, Instituto Polite´cnico Nacional, Me´xico, D.F., Me´xico. 3 Departamento de Ingenierı´a Quı´mica y Bioquı´mica, Instituto Tecnolo´gico de Tuxtepec, Tuxtepec, Me´xico. 2
ABSTRACT The involvement of angiotensin-I-converting enzyme (ACE-I) as one of the mechanisms controlling blood pressure is being studied to find alternative means of control of hypertension on human beings. On the market there are synthetic drugs that can control it, but these can cause undesirable health side effects. In this work was assessed the fractionation by ultrafiltration of the Lima bean (Phaseolus lunatus) and Jamapa bean (Phaseolus vulgaris), protein hydrolysates obtained with Alcalase and Flavourzyme on ACE-I inhibitory activity. Four membranes of different molecular cutoffs (10, 5, 3, and 1 kDa) were used. Fractions that had a higher inhibitory activity in both legumes were denominated as E ( < 1 kDa) with IC50 of 30.3 and 51.8 lg/mL values for the P. lunatus with Alcalase and Flavourzyme, respectively, and for the Phaseolus vulgaris with Alcalase and Flavourzyme with about 63.8 and 65.8 lg/mL values, respectively. The amino acid composition of these fractions showed residues in essential amino acids, which make a good source of energy and amino acids. On the other hand, the presence of hydrophobic amino acids such as V and P is a determining factor in the ACE-I inhibitor effect. The results suggest the possibility of obtaining and utilizing these peptide fractions in the development and innovation of a functional product that helps with treatment and/or prevention of hypertension.
KEY WORDS: ACE-I inhibitors peptide fractions Phaseolus lunatus Phaseolus vulgaris ultrafiltration
supported by FDA 221-02-3003. In response to these inherent risks, research interests have begun to focus more on food as a natural nonconventional source of ACE-I inhibitory peptides.6 Natural ACE-I inhibitor peptides are a promising alternative treatment because they do not produce side effects, although they are less potent.7 Bioactive peptides with the ACE-I inhibitory activity have been discovered in enzymatic hydrolysates of different food proteins, such as small red bean,8 soy,9 defatted canola meal,10 common bean (Phaseolus vulgaris L.),6 and they could be applied in the prevention of hypertension and in the initial treatment of mildly hypertensive individuals.11 This activity makes vegetables attractive sources for bioactive peptides extraction, particularly in the industrial sector (food and pharmaceutical) where peptides with antihypertensive activity can be added to food or drug systems. The potency of these antihypertensive peptides in vitro is expressed as an IC50 value, which is the ACE-I inhibitor concentration leading to 50% inhibition of ACE-I activity. Ultrafiltration (UF) using lowmolecular-weight cutoff (MWCO) membranes has been found useful in separating out high-molecular-weight residues from fractions rich in biologically active small peptides.12 The present work aims to evaluate the peptide fractions’ ACE-I inhibitory activity from the Lima bean (Phaseolus lunatus) and Jamapa bean (Phaseolus vulgaris) obtained by UF.
INTRODUCTION
T
he angiotensin-I-converting enzyme (ACE-I) (EC 3.4.15.1) plays a crucial role in the regulation of blood pressure as it promotes the conversion of angiotensin-I (DRVYIHPFHL) to the potent vasoconstrictor angiotensinII (DRVYIHPF) as well as inactivating the vasodilator bradykinin (RPPGFSPFR) by converting it into an inactive heptapeptide (RPPGFSP),1 and stimulates the release of aldosterone, causing sodium and fluid retention.2 Since then, many attempts have been made to synthesize ACE-I inhibitors such as Captopril, Enalapril/Enalaprilat, Quinapril, and Lisinopril, which are currently used in the treatment of essential hypertension and heart failure in humans.3 However, synthetic drugs produce side effects such as cough, taste alterations, skin rashes, or angioneurotic edema.4 On the other hand, the use of a number of ACE-I drugs during the first trimester of pregnancy is associated with increased risk of congenital malformations in the newborn5;
Manuscript received 9 January 2015. Revision accepted 12 April 2015. Address correspondence to: Dr. Juan Gabriel Torruco-Uco, Departamento de Ingenierı´a Quı´mica y Bioquı´mica, Instituto Tecnolo´gico de Tuxtepec, Av. Dr. Vı´ctor Bravo Ahuja S/N, C.P. 68350, Col. 5 de Mayo, Tuxtepec, Oaxaca, Me´xico, E-mail: [email protected]; [email protected]
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MATERIALS AND METHODS Materials Lima bean (P. lunatus) seeds were purchased in a market in Merida, Yucatan, Mexico, and Jamapa bean (Phaseolus vulgaris) seeds were donated by the Campo Experimental Valle de Me´xico (CEVAMEX), Texcoco, Estado de Mexico, Mexico. Reagents were analytical grade and purchased from J.T. Baker (Phillipsburg, NJ, USA), Sigma (Sigma Chemical Co., St. Louis, MO, USA), Merck (Darmstadt, Germany), and Bio-Rad (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Enzymes Alcalase 2.4 L FG and Flavourzyme 500 MG were purchased from Novo Laboratories (Copenhagen, Denmark). Protein concentrate The Lima bean (P. lunatus) and Jamapa bean (P. vulgaris) seeds were cleaned manually to eliminate impurities. After cleaning, seeds were ground in a Mykros impact mill until passing through a 20-mesh screen (0.85 mm) and then ground in a Cyclotec 1093 (Foss Tecator, Ho¨gana¨s, Sweden) mill until passing through a 60-mesh screen (0.24 mm). The resulting flour was mixed with water (1g:6mL), its pH adjusted to 11 with 1 N NaOH, and then soaked for 1 h with a mechanical shaker (Caframo RZ-1; Heidolph, Schwabach, Germany) at 5.18 g. This suspension was milled in a disk mill and passed through 80-mesh (0.19 mm) and 100-mesh (0.14 mm) screens to separate the fiber-containing solid fraction from the liquid fraction, which contains protein and starch. Residual solids were washed thrice using a 1g:3mL solid to distilled water ratio; the filtrate was collected in a plastic recipient and stored for 30 min at room temperature. Residues were dried at 60C in a convection oven (Lab-Line). The supernatant, containing starch-rich sediment, was decanted and dried at 60C in a convection oven (Lab-Line). Supernatant (solubilized proteins) pH was adjusted to their isoelectric point at 4.5 with 1 N HCl, and the suspension was centrifuged at 1317 g for 12 min using a Mistral 3000i centrifuge (Curtin Matheson Sci., Houston, TX, USA). The supernatants were discarded and the precipitates freeze-dried at - 47C and 13 · 10 - 3 mbar.13
electrode. Hydrolysis was stopped by heating to 85C for 15 min. The protein hydrolysates were clarified by filtering through 0.45-nm filters to remove the insoluble substrate and residual enzyme. The degree of hydrolysis (DH) was measured. Degree of hydrolysis DH was determined using the method of Kim et al.15 This value was estimated by measuring the soluble nitrogen content in 10 g trichloroacetic acid/100 mL of distilled water and determining its proportion versus total nitrogen concentration in the protein concentrate suspension according to the following equation: % DH ¼
10 g TCA/100 mL Soluble N · 100 Total N
Peptide fractions by UF The Lima bean (P. lunatus) and Jamapa bean (P. vulgaris) hydrolysates obtained with Alcalase and Flavourzyme were fractionated by UF according to Cho et al.16 using a highperformance UF cell (Model 2000; Millipore, Darmstadt, Germany). Five fractions were prepared using four MWCO membranes: 1K, 3K, 5K, and 10K. Soluble fractions were prepared by centrifuging the hydrolysates through the MWCO membranes beginning with the largest cartridge (10K). The retentate and permeate were collected separately, and the retentate was recirculated into the feed until the maximum permeate yield was reached, as indicated by a decreased permeate flow rate. Permeate from the 10K membrane was then filtered through the 5K membrane with recirculation until maximum permeate yield was reached. The 5K permeate was then processed with the 3K membrane and the 3K permeate with the 1K membrane. This process minimized contamination of the larger molecular weight fractions with smaller molecular weight fractions, while producing enough retentates and permeates for the following analysis. The five ultrafiltered peptide fractions were prepared and designated as fraction A: > 10K (10K retentate); fraction B: 5–10K (10K permeate–5K retentate); fraction C: 3–5K (5K permeate–3K retentate); fraction D: 1–3K (3K permeate–1K retentate); and fraction E: < 1K (1K permeate).
Enzymatic hydrolysis Protein concentrate hydrolysis was done using the method of Pedroche et al.14 by individual treatments with Alcalase 2.4L FG and Flavourzyme 500 MG (Novo Nordisk, Bagsvaerd, Denmark). Hydrolysis reaction times were according to previous research by Torruco-Uco et al.13 The P. lunatus and P. vulgaris, protein hydrolysates, were produced with Alcalase at 45 and 30 min, respectively, and with Flavourzyme in 90 min for both protein concentrates. The hydrolysis was done using the following parameters: substrate concentration 10 g/100 mL; enzyme/substrate ratio 0.3 AU/g for Alcalase and 50 LAPU/g for Flavourzyme; pH 8 for Alcalase and pH 7 for Flavourzyme; and 50C temperature for both. The hydrolysis was done in a 1000-mL beaker, equipped with a magnetic stirrer, thermometer, and pH
ACE-I inhibitory activity The ACE-I inhibitory activity in the hydrolysates and their UF peptide fractions was analyzed following Hayakari et al.17 In an ACE-I-containing hydrolysate, hippuryl-L-histidyl-Lleucine (HHL) yields hippuric acid and histidyl-leucine. This method is based on the colorimetric reaction of the hippuric acid with 2,4,6-trichloro-s-triazine (TT) in a 0.5-mL incubation mixture containing 40 lM of potassium phosphate buffer (pH 8.3), 300 lM of sodium chloride (pH 8.3), 3 g/100 mL of HHL in a potassium phosphate buffer (40 lM, pH 8.3), and 100 mU/mL of ACE-I. The mixture was incubated at 37C for 45 min and the reaction terminated by adding 3 g/100 mL of TT in dioxane and 3 mL of potassium phosphate buffer (0.2 M/L, pH 8.3). After centrifuging the reaction mixture at
ACE-I INHIBITORY PEPTIDE FRACTIONS FROM LEGUME SEEDS
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10,000 g for 10 min, the enzymatic activity was determined in the supernatant by measuring absorbance at 382 nm. The ACE-I inhibitory activity was determined by regression analysis of ACE-I inhibition (%) versus peptide concentration and defined as the absolute IC50 value, that is, the peptide concentration in mg protein/mL required to produce 50% ACE-I inhibition under the described conditions. Peptide fractions amino acid composition The peptide fractions amino acid composition showed a higher ACE-I inhibitory activity and it was determined following the method of Alaiz et al.18 Samples (2–4 mg protein) were treated with 4 mL HCl 6 M/L, placed in hydrolysis tubes, and gassed with nitrogen at 110C for 24 h. They were then dried in a rotavapor and suspended in a 1 M/ L sodium borate buffer at pH 9.0. Amino acid derivatization was done at 50C using diethyl ethoxymethylenemalonate. Amino acids were separated using HPLC with a reversed phase column (300 · 3.9 mm, Nova Pack C18, 4 lm; Waters), a binary gradient system with 25 mM/L sodium acetate containing (A) 0.02 g/L sodium azide at pH 6.0, and (B) acetonitrile as a solvent. The flow rate was 0.9 mL for a minute, and the elution gradient was as follows: time 0.0– 3.0 min, linear gradient A:B (91:9) to A–B (86:14); time 3.0–13.0 min, elution with A–B (86–14); time 13.0– 30.0 min, linear gradient A–B (86:14) to A-B (69:31); time 30.0–35.0 min, elution with A–B (69:31). Statistical analyses All results were analyzed using descriptive statistics with a central tendency and dispersion measures. One-way analysis of variance was applied to evaluate protein concentrate hydrolysis data and in vitro ACE-I inhibitory activity, and a Duncan’s multiple range test was used to determine differences between treatments. All analyses were done according to Montgomery,19 using the STATGRAPHICS Plus Version 5.1 software.
FIG. 1. Degree of hydrolysis (% DH) of the Phaseolus lunatus and Phaseolus vulgaris protein hydrolysates with Alcalase and Flavourzyme. Different lowercase letters (a–d) indicate statistical difference (P < .05).
in the hydrolysates produced by Alcalase (lunatus at 90 min IC50 = 0.056 mg/mL and vulgaris at 60 min IC50 = 0.061 mg/ mL), whereas the P. lunatus hydrolysate produced with Flavourzyme at 90 min had the highest inhibitory activity (IC50 = 0.0069 mg/mL); on the contrary, the P. vulgaris hydrolysate produced with Flavourzyme at 45 min had the lowest inhibitory activity (IC50 = 0.127 mg/mL) (Fig. 2). For the purpose of comparison, the IC50 value for captopril is 0.022 lg/mL.21 Then, as an alternative to the drug, all the above hydrolysates obtained showed acceptable ACE-I inhibitory activities, underwent UF, and their peptide fractions were analyzed. ACE-I fraction inhibitory activity of peptides by UF In all cases, except for fraction < 1 kDa (E), the ACEI inhibitory activity was statistically different (P < .05) between the same molecular size and different enzymatic system (Fig. 3). That E fraction with the lowest molecular weight increased its activity obtaining a value of IC50 = 30.3
RESULTS Enzymatic hydrolysis The highest DH for the P. lunatus and P. vulgaris protein concentrates was 37.94% and 49.48% produced with Alcalase at 45- and 30-min reaction time, respectively, than obtained with Flavourzyme with 22.03% and 26.05%, respectively, both to 90-min reactions (Fig. 1). In all cases, the values were statically different (P < .05). The DH level is commonly used as a parameter for monitoring the proteolysis and is the most common indicator of the hydrolyzed products.20 ACE-I inhibitory activity of protein hydrolysates. The ACE-I inhibitory activity of the P. lunatus y P. vulgaris hydrolysates was measured and calculated as IC50. It is understood that when the IC50 values are minors, the ACE-I inhibitory activity will be better. The ACE-I inhibitory activity in both Phaseolus was statistically the same (P > .05)
FIG. 2. ACE-I inhibitory activity (IC50 values) of the P. lunatus and P. vulgaris protein hydrolysates with Alcalase and Flavourzyme. Different lowercase letters (a–c) indicate statistical difference (P < .05). ACE-I, angiotensin-I-converting enzyme.
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tide fractions of lower molecular weight in a range of 294 to 63.8 lg/mL. Moreover, the peptide fractions that obtained hydrolysate with Flavourzyme P. vulgaris showed decreased ACE-I inhibitory activity in the first three major peptide fractions with an IC50 range of 228–326 lg/mL; then, this activity improved in fractions D (1–3 kDa) and E ( < 1 kDa) with an IC50 of 72.9 and 65.8 lg/mL, respectively, which were statistically equals (P > .05). Amino acid composition
FIG. 3. ACE-I inhibitory activity in vitro of P. lunatus peptides fractions with Alcalase and Flavourzyme. Different lowercase letters (a, b) in each figure indicate statistical difference (P < .05).
lg/mL opposite the P. lunatus Alcalase hydrolysate fraction with the lowest activity that was greatest for fraction > 10 kDa (A) with an IC50 = 443 lg/mL (Fig. 3). Furthermore, treatment with the P. lunatus Flavourzyme in fraction A ( > 10 kDa), an ACE-I inhibitory activity, was presented with an IC50 value = 17.5 lg/mL, and the activity decreased in fraction 5–10 kDa (B) with an IC50 = 601.5 lg/mL, and then this activity increased in the fraction < 1 kDa with an IC50 value = 51.8 lg/mL. The influence of enzymatic systems under P. vulgaris was slightly different since 1–3 and < 1 kDa fractions had the same values of ACE-I inhibitory activity (P > .05), as shown in Figure 4, and it can be seen that Alcalase has the same behavior as in P. lunatus with the same enzyme, since fraction A ( > 10 kDa) had the lowest ACE-I inhibitory activity with an IC50 = 387.5 lg/mL; however, this activity was improving in the following pep-
The amino acid composition of the peptide fractions with molecular weight < 1 kDa, which showed the highest ACE-I inhibitory activity (lower IC50) of legumes evaluated (P. lunatus and P. vulgaris), is shown in Table 1. The fractions showed high content of essential amino acids, such as Trp, Thr, Val, Phe, Tyr, Ile, Leu, Lys, Met, Cys, and His, being the sulfur amino acids and His the lowest value (P < .05) in the P. vulgaris Flavourzyme fraction. Then, the other fractions have a good balance in the amino acid composition as a source of protein in human nutrition.22 DISCUSSION The DH hydrolysates obtained for P. lunatus and P. vulgaris with the Alcalase enzyme were higher than reported in the protein hydrolysate obtained from protein isolated from Jatropha curcas with Alcalase with a value of Table 1. Amino Acid Composition of the Peptide Fractions ( < 1 kDa) Obtained from Phaseolus lunatus and Phaseolus vulgaris Protein Hydrolysates with Alcalase and Flavourzyme (%) FAO/ WHO/ P. lunatus P. lunatus P. vulgaris P. vulgaris ONU, Alcalase Flavourzyme Alcalase Flavourzyme 2002 Fractions < 1 kDa
Amino Acids
Asp + Asn 5.00b Glu + Gln 11.15a Ser 8.79b His* 4.23b Gly 5.15b,c Thr* 5.16a,b Arg 6.42a Ala 7.27b Pro 0.85a Val* 5.43a Met* 2.74c Cys* 0.46d Ile* 3.89a Leu* 10.86a Phe* 7.47a Tyr* 5.86a Lys* 7.39b Trp* 1.88b
FIG. 4. ACE-I inhibitory activity in vitro of P. vulgaris peptide fractions with Alcalase and Flavourzyme. Different lowercase letters (a, b) in each figure indicate statistical difference (P < .05).
3.94a 9.91a 5.08a 3.83b 4.28c 5.44a,b 8.08b 6.76a,b 0.70a 8.25b 2.71b,c 0.22b 6.48c 11.18a 8.48a 6.14a 7.37b 1.14a
Fractions < 1 kDa
4.21a,b 10.76a 10.07b 3.86b 5.18b 6.05b 6.03a 7.06b 0.43a 6.02a 2.24a,b 0.33c 4.04a,b 10.98a 8.39a 6.24a 7.00a,b 1.12a
6.50c 10.00a 6.80c 2.60a 3.24a 4.74a 6.68a 5.48a 10.18b 6.59a 1.87a 0.13a 4.91b 10.29a 7.97a 5.16a 5.76a 1.11a
1.5 2.3
3.9 2.2{ 3.0 5.9 3.8{{ 4.5 0.6
*Essential Amino Acids (FAO/WHO/ONU, 2002). { Met + Cys. {{ Phe + Tyr. Different superscript lowercase letters in the same row indicate statistical difference (P < .05).
ACE-I INHIBITORY PEPTIDE FRACTIONS FROM LEGUME SEEDS
13.9% at the 90-min reaction.23 Similarly, DH values were obtained by Hong et al.24 in protein hydrolysates; mung bean (Phaseolus radiatus L.) using Neutrase and Alcalase had values of 12% and 22%, respectively, and these were lower than those obtained in this study. Furthermore, Radha et al.25 using a combination of two enzymes (protease P ‘‘Amano’’ 6 and papain) obtained DH = 40% from a blend of flours from three oilseeds (soybeans, peanuts, sesame). The variability of these results may be due to the specificity of the enzyme used (Alcalase), which is an industrial alkaline protease from Bacillus licheniformis, where the main component of the enzyme is a serine endopeptidase Subtilisin Carlsberg and has a broad specificity for hydrolyzing peptide links from the C-terminal end, releasing peptides containing hydrophobic amino acid residues such as Phe (F), Tyr (Y), Trp (W), Leu (L), Ile (I), Val (V), and Met (M)13; therefore, the use of Alcalase enzyme is suitable for the production of peptides that inhibit ACE-I activity from protein concentrates of P. vulgaris and P. lunatus. Furthermore, hydrolysis obtained with protein hydrolysates from Flavourzyme in P. vulgaris and P. lunatus (22.03% and 26.05%, respectively) at the 90-min reaction was slower compared to the Alcalase enzyme, but larger to those found by Rui et al.26 Alcalase protein hydrolysates were obtained from protein isolates of three Phaseolus vulgaris varieties, namely, navy bean, black bean, and small red bean with DH 17.11%, 15.39%, and 13.90%, respectively, at the 90-min reaction. Other authors such as SeguraCampos et al.27 obtained 58.8% in DH protein hydrolysates of cowpea (Vigna unguiculata) after 90 min of reaction, which were higher than those found in this study. The hydrolytic behavior can be attributed to the different availability of susceptible peptide links, probably due to differences in the size and structure of the polypeptide chain comprising the proteins of legumes studied.20 It is equally noteworthy that the Flavourzyme enzyme is a mixture of endoproteases and exopeptidases and therefore can produce both free amino acids and peptides.28 The exopeptidase activity of this enzyme hydrolyzes peptide links of amino acids found at the end of the peptide chain, while the endoprotease hydrolyzes peptide bonds that are within the range producing a polypeptide chain, which may differ in molecular weight, depending on the extent of hydrolysis.29 Therefore, it can be deduced that Flavourzyme is affecting the peptide bonds, which are hydrophobic amino acid residues (mainly aromatic amino acids such as Trp, Phe, and Tyr), and proteins assessed by enzymatic specificity, which is less than the Alcalase enzyme, thus obtaining minor DH. The findings of this study were similar to the reported values to researchers in other vegetable sources; for example, the IC50 values found by Wu et al.10 obtained from enzymatic hydrolysates of Alcalase-treated defatted canola meal with and without heat treatment, showing an IC50 range from 0.0271 to 0.0443 mg/mL. Similarly, Wu et al.30 using the same enzyme (Alcalase) indicated IC50 = 0.0250 mg/mL in hydrolysates from defatted canola meal treated with ethanol and an IC50 value = 0.0286 mg/mL in the same flour without ethanol treatment.
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Furthermore, Segura-Campos et al.27 found IC50 = 2.6344 mg/mL protein hydrolysates in cowpea (Vigna unguiculata) obtained from Flavourzyme. In addition, Choi et al.31 reported IC50 values greater than = 0.95, 3.5, and 4.2 mg/mL of the extract obtained from nuts of three varieties of mushrooms (Grifola frondosa, Coriolus versicolor, and Pleurotus coccinea, respectively) suspended in cold water. Chen et al.32 found IC50 = 0.460 mg/mL in rice protein hydrolysates obtained by sequential enzymatic hydrolysis with Alcalase and Trypsin. Whereas Suh et al.33 from protein hydrolysates of wheat gluten obtained ACE-I inhibitory activity with IC50 values from 0.16 to 0.18 mg/mL, using the enzyme Flavourzyme during 6 and 8 h of hydrolysis, respectively; these values were similar to those found in this study with the same enzyme. Other researchers such as Pedroche et al.14 found values of ACE-I inhibition in the pea protein isolate, with IC50 = 0.19 mg/mL, being sequentially hydrolyzed with Alcalase and Flavourzyme. Previous studies have reported that Alcalase is capable of producing bioactive peptides when it is used to hydrolyze protein foods.34 Furthermore, the sequence produces Alcalase shorter peptides as well as terminal amino acid sequences responsible for several health beneficial bioactivities, including ACE-I inhibition. Besides, bioactive peptides produced by this protease are resistant to digestive enzymes such as pepsin, trypsin, and chymotrypsin, which may allow the absorption of peptides contained in the hydrolysate by the small intestine as it has been shown by various studies in spontaneously hypertensive rats.9,35 UF is commonly used to isolate ACE-I inhibitory peptides from food proteins.32 The results obtained in this research are comparable to those obtained by Kamath et al.36 and from them the main a-kafirin storage protein sorghum (Sorghum bicolor) was obtained; peptide fractions using column gel filtration (Sephadex G-25) and the IC50 values found from 1.3 to 24.3 lg/mL were similar to the values found in legumes studied by UF. Furthermore, Jung et al.37 obtained from fish skeleton yellowfin sole (Limanda aspera) protein hydrolysates with a-chymotrypsin that were fractionated in different molecular cuts membranes: 30, 10 and 5 kDa, finding a value of IC50 = 883 lg/mL in the fraction < 5 kDa, which had a lower ACE-I inhibitory activity than the values found in this study. These showed better IC50 values in the peptide fractions with a molecular < 5 kDa weight in both legumes evaluated. Other researchers such as Chen et al.32 protein hydrolysates obtained from rice using the enzymes Alcalase and Trypsin and then these were fractionated by UF using two membranes of different molecular cut (3 to 10 kDa), and the values of IC50 were found in the fraction > 10 kDa = 1570 lg/mL, 3–10 kDa fraction = 840 lg/mL, and the fraction < 3 kDa = 280 lg/mL; the latter value is comparable with the results obtained in this study as fraction D obtained in 1– 3 kDa of P. lunatus hydrolyzed with Alcalase presented in IC50 = 262.5 lg/mL, which had better activity. These results are consistent with other reports of other investigators since ACE-I inhibitory activity increases as the molecular weight
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of the peptide fractions decreases.12 Likewise, these differences in IC50 values could be due to the amino acid composition and sequence of the same; since each fraction showed that IC50 values were different, this feature will depend on the aforementioned factors. It is noteworthy that these amino acids have been previously found in other lupin protein hydrolysates,38 buckwheat,39 and azufrado beans (Phaseolus vulgaris).40 Furthermore, the presence of hydrophobic amino acids in peptide fractions of legumes evaluated showed high in V (54.3–82.5 g kg - 1), I (38.9–64.8 g kg - 1), P (4.3–101.8 g kg - 1), Met (18.7–27.4 g kg - 1), F (74.7–84.8 g kg - 1), L (102.9– 111.8 g kg - 1), and W (11.1–18.8 g kg - 1), and these amino acids are found in the amino acid sequence of different peptide fractions obtained from protein hydrolysates, as ‘‘GPP’’ for buckwheat,41 ‘‘LAIPVNSP’’ for soya,42 ‘‘VPP’’ for milk,43 ‘‘IFVPAF’’ for sea shrimp,44 and ‘‘IGDEPLANYL’’ for jellyfish.45 The ACE-I inhibitory activities imply that the hydrophobic amino acids may be present within the amino acid sequence of peptide fractions ( < 1 kDa) obtained from hydrolysates and P. vulgaris and P. lunatus with enzymes Alcalase and Flavourzyme, as they showed IC50 values of 30.3, 51.8, 63.8, and 65.8 lg/mL, respectively, on the ACE-I inhibition. Other researchers such as Ohba et al.46 reported P amino acid residues within the amino acid sequence of peptides found in the peptide fractions obtained ( < 1 kDa) from hydrolyzed collagen or keratin livestock and fish waste (bone and flesh), and these showed ACE-I inhibitory activity with an IC50 between 600 and 2800 lg/mL. Jung et al.37 found in the protein hydrolysate from skeleton Limanda aspera, the presence of hydrophobic amino acid residues within the amino acid sequence of a peptide, MIFPGAGGPEL, with a molecular weight of 1.3 kDa, and it showed ACE-I inhibitory activity with an IC50 = 28.7 lg/mL, which was similar to the values found in this study. Therefore, it has been recognized that the amino acid composition of the ACE-I inhibitory peptides is critical to possess inhibitory activity. Similarly, the structure–activity relationship indicates that the bond with ACE-I can be strongly affected by the tripeptide sequence in the Cterminal of the substrate and it is proposed that peptides include hydrophobic amino acids at these positions that are potent inhibitors of ACE-I.47 Therefore, our results suggest that ACE-I-inhibiting peptides found in peptide fractions < 1 kDa P. vulgaris and P. lunatus may contain hydrophobic amino acid residues in the C-terminal residues, especially P and A. Therefore, these results demonstrate to be important the structural identification of ACE-I inhibitory peptides in fractions of P. vulgaris and P. Iunatus. Since the primary activity of ACE-I is to cleave the Cterminal dipeptide of oligopeptide substrates with a wide specificity, the inhibitory activity of ACE-I inhibitory peptides is strongly influenced by their C-terminal tripeptide sequence. The most potent ACE-I inhibitors contain hydrophobic amino acid residues at each of the three Cterminal positions that interact with the subsites S1, S19, and S29 at the ACE-I active site.
Many studies have shown that peptides with high ACE-I inhibitory activities have tryptophan, phenylalanine, tyrosine, or proline at their C-terminus and branched aliphatic amino acids at the N-terminus. According to Cheung et al.,48 ACE-I is known to have little affinity for substrates or competitive inhibitor mechanisms with COOH-terminal dicarboxylic amino acids or with penultimate proline residues, but an antepenultimate aromatic amino acid residue appears to enhance binding. Bradykinin (Arg-Pro-Gly-PheSer-Pro-Phe-Arg) is bound more tightly to the enzyme than angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) and the pentapeptide substrate inhibitor; BPP is bound more tightly than either of these. In conclusion, protein hydrolysates, P. lunatus and P. vulgaris, obtained by enzymatic hydrolysis with Alcalase and Flavourzyme generated ACE-I inhibitory activity. Fractionation of these hydrolysates by UF originated peptide fractions of low-molecular weight ( < 1 kDa) that had a greater activity. Therefore, these fractions may have the commercial potential to be used as a functional ingredient for the prevention and/or treatment of hypertension.
ACKNOWLEDGMENTS This research was partially funded by the Consejo Nacional de Ciencia y Tecnologı´a (CONACYT) through doctoral scholarship 43867 and Project Ciencia Ba´sica 153012, the SIP-IPN (projects: 20060445, 20070800, and 20082532), and a scholarship from the Programa Institucional de Formacio´n de Investigadores (PIFI). AUTHOR DISCLOSURE STATEMENT No competing financial interests exist. REFERENCES 1. Chen YH, Liu YH, Yang YH, Feng HH, Chang CT, Chen CC: Antihypertensive effect of an enzymatic hydrolysate of chicken essence residues. Food Sci Technol Res 2002;8:144–147. 2. Scow TD, Smith GE, Shaughnessy FA: Combination therapy with ACE inhibitors and angiotensin-receptor blockers in heart failure. Clin Pharmacol 2003;68:1795–1798. 3. Hatanaka A, Miyahara H, Suzuki KI, Sato S: Isolation and identification of antihypertensive peptides from Antarctic krill tail meat hydrolysate. J Food Sci 2009;74:H116–H120. 4. Chiang WD, Tsou MJ, Tsai ZY, Tsai TC: Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor. Food Chem 2006; 98:725–732. 5. Copper OW, Herna´ndez-Diaz S, Arbogast GP, et al.: Major congenital malformations after first-trimester exposure to ACE inhibitors. N Engl J Med 2006;34:2443–2451. 6. Luna-Vital DA, Mojica L, Gonza´lez de Mejı´a E, Mendoza S, Loarca-Pin˜a G: Biological potential of protein hydrolysates and peptides from common bean (Phaseolus vulgaris L.): A review. Food Res Int [Epub ahead of print]; DOI: 10.1016/j.foodres .2014.11.024. 7. Cao W, Zhang C, Hong P, Ji H, Hao J: Purification and identification of an ACE inhibitory peptide from the peptic hydroly-
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FULL COMMUNICATION
ACE-I Inhibitory Activity from Phaseolus lunatus and Phaseolus vulgaris Peptide Fractions Obtained by Ultrafiltration David Betancur-Ancona,1 Gloria Da´vila-Ortiz,2 Luis Antonio Chel-Guerrero,1 and Juan Gabriel Torruco-Uco 2,3 1
Facultad de Ingenierı´a Quı´mica, Universidad Auto´noma de Yucata´n, Me´rida, Me´xico. Escuela Nacional de Ciencias Biolo´gicas, Instituto Polite´cnico Nacional, Me´xico, D.F., Me´xico. 3 Departamento de Ingenierı´a Quı´mica y Bioquı´mica, Instituto Tecnolo´gico de Tuxtepec, Tuxtepec, Me´xico. 2
ABSTRACT The involvement of angiotensin-I-converting enzyme (ACE-I) as one of the mechanisms controlling blood pressure is being studied to find alternative means of control of hypertension on human beings. On the market there are synthetic drugs that can control it, but these can cause undesirable health side effects. In this work was assessed the fractionation by ultrafiltration of the Lima bean (Phaseolus lunatus) and Jamapa bean (Phaseolus vulgaris), protein hydrolysates obtained with Alcalase and Flavourzyme on ACE-I inhibitory activity. Four membranes of different molecular cutoffs (10, 5, 3, and 1 kDa) were used. Fractions that had a higher inhibitory activity in both legumes were denominated as E ( < 1 kDa) with IC50 of 30.3 and 51.8 lg/mL values for the P. lunatus with Alcalase and Flavourzyme, respectively, and for the Phaseolus vulgaris with Alcalase and Flavourzyme with about 63.8 and 65.8 lg/mL values, respectively. The amino acid composition of these fractions showed residues in essential amino acids, which make a good source of energy and amino acids. On the other hand, the presence of hydrophobic amino acids such as V and P is a determining factor in the ACE-I inhibitor effect. The results suggest the possibility of obtaining and utilizing these peptide fractions in the development and innovation of a functional product that helps with treatment and/or prevention of hypertension.
KEY WORDS: ACE-I inhibitors peptide fractions Phaseolus lunatus Phaseolus vulgaris ultrafiltration
supported by FDA 221-02-3003. In response to these inherent risks, research interests have begun to focus more on food as a natural nonconventional source of ACE-I inhibitory peptides.6 Natural ACE-I inhibitor peptides are a promising alternative treatment because they do not produce side effects, although they are less potent.7 Bioactive peptides with the ACE-I inhibitory activity have been discovered in enzymatic hydrolysates of different food proteins, such as small red bean,8 soy,9 defatted canola meal,10 common bean (Phaseolus vulgaris L.),6 and they could be applied in the prevention of hypertension and in the initial treatment of mildly hypertensive individuals.11 This activity makes vegetables attractive sources for bioactive peptides extraction, particularly in the industrial sector (food and pharmaceutical) where peptides with antihypertensive activity can be added to food or drug systems. The potency of these antihypertensive peptides in vitro is expressed as an IC50 value, which is the ACE-I inhibitor concentration leading to 50% inhibition of ACE-I activity. Ultrafiltration (UF) using lowmolecular-weight cutoff (MWCO) membranes has been found useful in separating out high-molecular-weight residues from fractions rich in biologically active small peptides.12 The present work aims to evaluate the peptide fractions’ ACE-I inhibitory activity from the Lima bean (Phaseolus lunatus) and Jamapa bean (Phaseolus vulgaris) obtained by UF.
INTRODUCTION
T
he angiotensin-I-converting enzyme (ACE-I) (EC 3.4.15.1) plays a crucial role in the regulation of blood pressure as it promotes the conversion of angiotensin-I (DRVYIHPFHL) to the potent vasoconstrictor angiotensinII (DRVYIHPF) as well as inactivating the vasodilator bradykinin (RPPGFSPFR) by converting it into an inactive heptapeptide (RPPGFSP),1 and stimulates the release of aldosterone, causing sodium and fluid retention.2 Since then, many attempts have been made to synthesize ACE-I inhibitors such as Captopril, Enalapril/Enalaprilat, Quinapril, and Lisinopril, which are currently used in the treatment of essential hypertension and heart failure in humans.3 However, synthetic drugs produce side effects such as cough, taste alterations, skin rashes, or angioneurotic edema.4 On the other hand, the use of a number of ACE-I drugs during the first trimester of pregnancy is associated with increased risk of congenital malformations in the newborn5;
Manuscript received 9 January 2015. Revision accepted 12 April 2015. Address correspondence to: Dr. Juan Gabriel Torruco-Uco, Departamento de Ingenierı´a Quı´mica y Bioquı´mica, Instituto Tecnolo´gico de Tuxtepec, Av. Dr. Vı´ctor Bravo Ahuja S/N, C.P. 68350, Col. 5 de Mayo, Tuxtepec, Oaxaca, Me´xico, E-mail: [email protected]; [email protected]
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MATERIALS AND METHODS Materials Lima bean (P. lunatus) seeds were purchased in a market in Merida, Yucatan, Mexico, and Jamapa bean (Phaseolus vulgaris) seeds were donated by the Campo Experimental Valle de Me´xico (CEVAMEX), Texcoco, Estado de Mexico, Mexico. Reagents were analytical grade and purchased from J.T. Baker (Phillipsburg, NJ, USA), Sigma (Sigma Chemical Co., St. Louis, MO, USA), Merck (Darmstadt, Germany), and Bio-Rad (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Enzymes Alcalase 2.4 L FG and Flavourzyme 500 MG were purchased from Novo Laboratories (Copenhagen, Denmark). Protein concentrate The Lima bean (P. lunatus) and Jamapa bean (P. vulgaris) seeds were cleaned manually to eliminate impurities. After cleaning, seeds were ground in a Mykros impact mill until passing through a 20-mesh screen (0.85 mm) and then ground in a Cyclotec 1093 (Foss Tecator, Ho¨gana¨s, Sweden) mill until passing through a 60-mesh screen (0.24 mm). The resulting flour was mixed with water (1g:6mL), its pH adjusted to 11 with 1 N NaOH, and then soaked for 1 h with a mechanical shaker (Caframo RZ-1; Heidolph, Schwabach, Germany) at 5.18 g. This suspension was milled in a disk mill and passed through 80-mesh (0.19 mm) and 100-mesh (0.14 mm) screens to separate the fiber-containing solid fraction from the liquid fraction, which contains protein and starch. Residual solids were washed thrice using a 1g:3mL solid to distilled water ratio; the filtrate was collected in a plastic recipient and stored for 30 min at room temperature. Residues were dried at 60C in a convection oven (Lab-Line). The supernatant, containing starch-rich sediment, was decanted and dried at 60C in a convection oven (Lab-Line). Supernatant (solubilized proteins) pH was adjusted to their isoelectric point at 4.5 with 1 N HCl, and the suspension was centrifuged at 1317 g for 12 min using a Mistral 3000i centrifuge (Curtin Matheson Sci., Houston, TX, USA). The supernatants were discarded and the precipitates freeze-dried at - 47C and 13 · 10 - 3 mbar.13
electrode. Hydrolysis was stopped by heating to 85C for 15 min. The protein hydrolysates were clarified by filtering through 0.45-nm filters to remove the insoluble substrate and residual enzyme. The degree of hydrolysis (DH) was measured. Degree of hydrolysis DH was determined using the method of Kim et al.15 This value was estimated by measuring the soluble nitrogen content in 10 g trichloroacetic acid/100 mL of distilled water and determining its proportion versus total nitrogen concentration in the protein concentrate suspension according to the following equation: % DH ¼
10 g TCA/100 mL Soluble N · 100 Total N
Peptide fractions by UF The Lima bean (P. lunatus) and Jamapa bean (P. vulgaris) hydrolysates obtained with Alcalase and Flavourzyme were fractionated by UF according to Cho et al.16 using a highperformance UF cell (Model 2000; Millipore, Darmstadt, Germany). Five fractions were prepared using four MWCO membranes: 1K, 3K, 5K, and 10K. Soluble fractions were prepared by centrifuging the hydrolysates through the MWCO membranes beginning with the largest cartridge (10K). The retentate and permeate were collected separately, and the retentate was recirculated into the feed until the maximum permeate yield was reached, as indicated by a decreased permeate flow rate. Permeate from the 10K membrane was then filtered through the 5K membrane with recirculation until maximum permeate yield was reached. The 5K permeate was then processed with the 3K membrane and the 3K permeate with the 1K membrane. This process minimized contamination of the larger molecular weight fractions with smaller molecular weight fractions, while producing enough retentates and permeates for the following analysis. The five ultrafiltered peptide fractions were prepared and designated as fraction A: > 10K (10K retentate); fraction B: 5–10K (10K permeate–5K retentate); fraction C: 3–5K (5K permeate–3K retentate); fraction D: 1–3K (3K permeate–1K retentate); and fraction E: < 1K (1K permeate).
Enzymatic hydrolysis Protein concentrate hydrolysis was done using the method of Pedroche et al.14 by individual treatments with Alcalase 2.4L FG and Flavourzyme 500 MG (Novo Nordisk, Bagsvaerd, Denmark). Hydrolysis reaction times were according to previous research by Torruco-Uco et al.13 The P. lunatus and P. vulgaris, protein hydrolysates, were produced with Alcalase at 45 and 30 min, respectively, and with Flavourzyme in 90 min for both protein concentrates. The hydrolysis was done using the following parameters: substrate concentration 10 g/100 mL; enzyme/substrate ratio 0.3 AU/g for Alcalase and 50 LAPU/g for Flavourzyme; pH 8 for Alcalase and pH 7 for Flavourzyme; and 50C temperature for both. The hydrolysis was done in a 1000-mL beaker, equipped with a magnetic stirrer, thermometer, and pH
ACE-I inhibitory activity The ACE-I inhibitory activity in the hydrolysates and their UF peptide fractions was analyzed following Hayakari et al.17 In an ACE-I-containing hydrolysate, hippuryl-L-histidyl-Lleucine (HHL) yields hippuric acid and histidyl-leucine. This method is based on the colorimetric reaction of the hippuric acid with 2,4,6-trichloro-s-triazine (TT) in a 0.5-mL incubation mixture containing 40 lM of potassium phosphate buffer (pH 8.3), 300 lM of sodium chloride (pH 8.3), 3 g/100 mL of HHL in a potassium phosphate buffer (40 lM, pH 8.3), and 100 mU/mL of ACE-I. The mixture was incubated at 37C for 45 min and the reaction terminated by adding 3 g/100 mL of TT in dioxane and 3 mL of potassium phosphate buffer (0.2 M/L, pH 8.3). After centrifuging the reaction mixture at
ACE-I INHIBITORY PEPTIDE FRACTIONS FROM LEGUME SEEDS
3
10,000 g for 10 min, the enzymatic activity was determined in the supernatant by measuring absorbance at 382 nm. The ACE-I inhibitory activity was determined by regression analysis of ACE-I inhibition (%) versus peptide concentration and defined as the absolute IC50 value, that is, the peptide concentration in mg protein/mL required to produce 50% ACE-I inhibition under the described conditions. Peptide fractions amino acid composition The peptide fractions amino acid composition showed a higher ACE-I inhibitory activity and it was determined following the method of Alaiz et al.18 Samples (2–4 mg protein) were treated with 4 mL HCl 6 M/L, placed in hydrolysis tubes, and gassed with nitrogen at 110C for 24 h. They were then dried in a rotavapor and suspended in a 1 M/ L sodium borate buffer at pH 9.0. Amino acid derivatization was done at 50C using diethyl ethoxymethylenemalonate. Amino acids were separated using HPLC with a reversed phase column (300 · 3.9 mm, Nova Pack C18, 4 lm; Waters), a binary gradient system with 25 mM/L sodium acetate containing (A) 0.02 g/L sodium azide at pH 6.0, and (B) acetonitrile as a solvent. The flow rate was 0.9 mL for a minute, and the elution gradient was as follows: time 0.0– 3.0 min, linear gradient A:B (91:9) to A–B (86:14); time 3.0–13.0 min, elution with A–B (86–14); time 13.0– 30.0 min, linear gradient A–B (86:14) to A-B (69:31); time 30.0–35.0 min, elution with A–B (69:31). Statistical analyses All results were analyzed using descriptive statistics with a central tendency and dispersion measures. One-way analysis of variance was applied to evaluate protein concentrate hydrolysis data and in vitro ACE-I inhibitory activity, and a Duncan’s multiple range test was used to determine differences between treatments. All analyses were done according to Montgomery,19 using the STATGRAPHICS Plus Version 5.1 software.
FIG. 1. Degree of hydrolysis (% DH) of the Phaseolus lunatus and Phaseolus vulgaris protein hydrolysates with Alcalase and Flavourzyme. Different lowercase letters (a–d) indicate statistical difference (P < .05).
in the hydrolysates produced by Alcalase (lunatus at 90 min IC50 = 0.056 mg/mL and vulgaris at 60 min IC50 = 0.061 mg/ mL), whereas the P. lunatus hydrolysate produced with Flavourzyme at 90 min had the highest inhibitory activity (IC50 = 0.0069 mg/mL); on the contrary, the P. vulgaris hydrolysate produced with Flavourzyme at 45 min had the lowest inhibitory activity (IC50 = 0.127 mg/mL) (Fig. 2). For the purpose of comparison, the IC50 value for captopril is 0.022 lg/mL.21 Then, as an alternative to the drug, all the above hydrolysates obtained showed acceptable ACE-I inhibitory activities, underwent UF, and their peptide fractions were analyzed. ACE-I fraction inhibitory activity of peptides by UF In all cases, except for fraction < 1 kDa (E), the ACEI inhibitory activity was statistically different (P < .05) between the same molecular size and different enzymatic system (Fig. 3). That E fraction with the lowest molecular weight increased its activity obtaining a value of IC50 = 30.3
RESULTS Enzymatic hydrolysis The highest DH for the P. lunatus and P. vulgaris protein concentrates was 37.94% and 49.48% produced with Alcalase at 45- and 30-min reaction time, respectively, than obtained with Flavourzyme with 22.03% and 26.05%, respectively, both to 90-min reactions (Fig. 1). In all cases, the values were statically different (P < .05). The DH level is commonly used as a parameter for monitoring the proteolysis and is the most common indicator of the hydrolyzed products.20 ACE-I inhibitory activity of protein hydrolysates. The ACE-I inhibitory activity of the P. lunatus y P. vulgaris hydrolysates was measured and calculated as IC50. It is understood that when the IC50 values are minors, the ACE-I inhibitory activity will be better. The ACE-I inhibitory activity in both Phaseolus was statistically the same (P > .05)
FIG. 2. ACE-I inhibitory activity (IC50 values) of the P. lunatus and P. vulgaris protein hydrolysates with Alcalase and Flavourzyme. Different lowercase letters (a–c) indicate statistical difference (P < .05). ACE-I, angiotensin-I-converting enzyme.
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tide fractions of lower molecular weight in a range of 294 to 63.8 lg/mL. Moreover, the peptide fractions that obtained hydrolysate with Flavourzyme P. vulgaris showed decreased ACE-I inhibitory activity in the first three major peptide fractions with an IC50 range of 228–326 lg/mL; then, this activity improved in fractions D (1–3 kDa) and E ( < 1 kDa) with an IC50 of 72.9 and 65.8 lg/mL, respectively, which were statistically equals (P > .05). Amino acid composition
FIG. 3. ACE-I inhibitory activity in vitro of P. lunatus peptides fractions with Alcalase and Flavourzyme. Different lowercase letters (a, b) in each figure indicate statistical difference (P < .05).
lg/mL opposite the P. lunatus Alcalase hydrolysate fraction with the lowest activity that was greatest for fraction > 10 kDa (A) with an IC50 = 443 lg/mL (Fig. 3). Furthermore, treatment with the P. lunatus Flavourzyme in fraction A ( > 10 kDa), an ACE-I inhibitory activity, was presented with an IC50 value = 17.5 lg/mL, and the activity decreased in fraction 5–10 kDa (B) with an IC50 = 601.5 lg/mL, and then this activity increased in the fraction < 1 kDa with an IC50 value = 51.8 lg/mL. The influence of enzymatic systems under P. vulgaris was slightly different since 1–3 and < 1 kDa fractions had the same values of ACE-I inhibitory activity (P > .05), as shown in Figure 4, and it can be seen that Alcalase has the same behavior as in P. lunatus with the same enzyme, since fraction A ( > 10 kDa) had the lowest ACE-I inhibitory activity with an IC50 = 387.5 lg/mL; however, this activity was improving in the following pep-
The amino acid composition of the peptide fractions with molecular weight < 1 kDa, which showed the highest ACE-I inhibitory activity (lower IC50) of legumes evaluated (P. lunatus and P. vulgaris), is shown in Table 1. The fractions showed high content of essential amino acids, such as Trp, Thr, Val, Phe, Tyr, Ile, Leu, Lys, Met, Cys, and His, being the sulfur amino acids and His the lowest value (P < .05) in the P. vulgaris Flavourzyme fraction. Then, the other fractions have a good balance in the amino acid composition as a source of protein in human nutrition.22 DISCUSSION The DH hydrolysates obtained for P. lunatus and P. vulgaris with the Alcalase enzyme were higher than reported in the protein hydrolysate obtained from protein isolated from Jatropha curcas with Alcalase with a value of Table 1. Amino Acid Composition of the Peptide Fractions ( < 1 kDa) Obtained from Phaseolus lunatus and Phaseolus vulgaris Protein Hydrolysates with Alcalase and Flavourzyme (%) FAO/ WHO/ P. lunatus P. lunatus P. vulgaris P. vulgaris ONU, Alcalase Flavourzyme Alcalase Flavourzyme 2002 Fractions < 1 kDa
Amino Acids
Asp + Asn 5.00b Glu + Gln 11.15a Ser 8.79b His* 4.23b Gly 5.15b,c Thr* 5.16a,b Arg 6.42a Ala 7.27b Pro 0.85a Val* 5.43a Met* 2.74c Cys* 0.46d Ile* 3.89a Leu* 10.86a Phe* 7.47a Tyr* 5.86a Lys* 7.39b Trp* 1.88b
FIG. 4. ACE-I inhibitory activity in vitro of P. vulgaris peptide fractions with Alcalase and Flavourzyme. Different lowercase letters (a, b) in each figure indicate statistical difference (P < .05).
3.94a 9.91a 5.08a 3.83b 4.28c 5.44a,b 8.08b 6.76a,b 0.70a 8.25b 2.71b,c 0.22b 6.48c 11.18a 8.48a 6.14a 7.37b 1.14a
Fractions < 1 kDa
4.21a,b 10.76a 10.07b 3.86b 5.18b 6.05b 6.03a 7.06b 0.43a 6.02a 2.24a,b 0.33c 4.04a,b 10.98a 8.39a 6.24a 7.00a,b 1.12a
6.50c 10.00a 6.80c 2.60a 3.24a 4.74a 6.68a 5.48a 10.18b 6.59a 1.87a 0.13a 4.91b 10.29a 7.97a 5.16a 5.76a 1.11a
1.5 2.3
3.9 2.2{ 3.0 5.9 3.8{{ 4.5 0.6
*Essential Amino Acids (FAO/WHO/ONU, 2002). { Met + Cys. {{ Phe + Tyr. Different superscript lowercase letters in the same row indicate statistical difference (P < .05).
ACE-I INHIBITORY PEPTIDE FRACTIONS FROM LEGUME SEEDS
13.9% at the 90-min reaction.23 Similarly, DH values were obtained by Hong et al.24 in protein hydrolysates; mung bean (Phaseolus radiatus L.) using Neutrase and Alcalase had values of 12% and 22%, respectively, and these were lower than those obtained in this study. Furthermore, Radha et al.25 using a combination of two enzymes (protease P ‘‘Amano’’ 6 and papain) obtained DH = 40% from a blend of flours from three oilseeds (soybeans, peanuts, sesame). The variability of these results may be due to the specificity of the enzyme used (Alcalase), which is an industrial alkaline protease from Bacillus licheniformis, where the main component of the enzyme is a serine endopeptidase Subtilisin Carlsberg and has a broad specificity for hydrolyzing peptide links from the C-terminal end, releasing peptides containing hydrophobic amino acid residues such as Phe (F), Tyr (Y), Trp (W), Leu (L), Ile (I), Val (V), and Met (M)13; therefore, the use of Alcalase enzyme is suitable for the production of peptides that inhibit ACE-I activity from protein concentrates of P. vulgaris and P. lunatus. Furthermore, hydrolysis obtained with protein hydrolysates from Flavourzyme in P. vulgaris and P. lunatus (22.03% and 26.05%, respectively) at the 90-min reaction was slower compared to the Alcalase enzyme, but larger to those found by Rui et al.26 Alcalase protein hydrolysates were obtained from protein isolates of three Phaseolus vulgaris varieties, namely, navy bean, black bean, and small red bean with DH 17.11%, 15.39%, and 13.90%, respectively, at the 90-min reaction. Other authors such as SeguraCampos et al.27 obtained 58.8% in DH protein hydrolysates of cowpea (Vigna unguiculata) after 90 min of reaction, which were higher than those found in this study. The hydrolytic behavior can be attributed to the different availability of susceptible peptide links, probably due to differences in the size and structure of the polypeptide chain comprising the proteins of legumes studied.20 It is equally noteworthy that the Flavourzyme enzyme is a mixture of endoproteases and exopeptidases and therefore can produce both free amino acids and peptides.28 The exopeptidase activity of this enzyme hydrolyzes peptide links of amino acids found at the end of the peptide chain, while the endoprotease hydrolyzes peptide bonds that are within the range producing a polypeptide chain, which may differ in molecular weight, depending on the extent of hydrolysis.29 Therefore, it can be deduced that Flavourzyme is affecting the peptide bonds, which are hydrophobic amino acid residues (mainly aromatic amino acids such as Trp, Phe, and Tyr), and proteins assessed by enzymatic specificity, which is less than the Alcalase enzyme, thus obtaining minor DH. The findings of this study were similar to the reported values to researchers in other vegetable sources; for example, the IC50 values found by Wu et al.10 obtained from enzymatic hydrolysates of Alcalase-treated defatted canola meal with and without heat treatment, showing an IC50 range from 0.0271 to 0.0443 mg/mL. Similarly, Wu et al.30 using the same enzyme (Alcalase) indicated IC50 = 0.0250 mg/mL in hydrolysates from defatted canola meal treated with ethanol and an IC50 value = 0.0286 mg/mL in the same flour without ethanol treatment.
5
Furthermore, Segura-Campos et al.27 found IC50 = 2.6344 mg/mL protein hydrolysates in cowpea (Vigna unguiculata) obtained from Flavourzyme. In addition, Choi et al.31 reported IC50 values greater than = 0.95, 3.5, and 4.2 mg/mL of the extract obtained from nuts of three varieties of mushrooms (Grifola frondosa, Coriolus versicolor, and Pleurotus coccinea, respectively) suspended in cold water. Chen et al.32 found IC50 = 0.460 mg/mL in rice protein hydrolysates obtained by sequential enzymatic hydrolysis with Alcalase and Trypsin. Whereas Suh et al.33 from protein hydrolysates of wheat gluten obtained ACE-I inhibitory activity with IC50 values from 0.16 to 0.18 mg/mL, using the enzyme Flavourzyme during 6 and 8 h of hydrolysis, respectively; these values were similar to those found in this study with the same enzyme. Other researchers such as Pedroche et al.14 found values of ACE-I inhibition in the pea protein isolate, with IC50 = 0.19 mg/mL, being sequentially hydrolyzed with Alcalase and Flavourzyme. Previous studies have reported that Alcalase is capable of producing bioactive peptides when it is used to hydrolyze protein foods.34 Furthermore, the sequence produces Alcalase shorter peptides as well as terminal amino acid sequences responsible for several health beneficial bioactivities, including ACE-I inhibition. Besides, bioactive peptides produced by this protease are resistant to digestive enzymes such as pepsin, trypsin, and chymotrypsin, which may allow the absorption of peptides contained in the hydrolysate by the small intestine as it has been shown by various studies in spontaneously hypertensive rats.9,35 UF is commonly used to isolate ACE-I inhibitory peptides from food proteins.32 The results obtained in this research are comparable to those obtained by Kamath et al.36 and from them the main a-kafirin storage protein sorghum (Sorghum bicolor) was obtained; peptide fractions using column gel filtration (Sephadex G-25) and the IC50 values found from 1.3 to 24.3 lg/mL were similar to the values found in legumes studied by UF. Furthermore, Jung et al.37 obtained from fish skeleton yellowfin sole (Limanda aspera) protein hydrolysates with a-chymotrypsin that were fractionated in different molecular cuts membranes: 30, 10 and 5 kDa, finding a value of IC50 = 883 lg/mL in the fraction < 5 kDa, which had a lower ACE-I inhibitory activity than the values found in this study. These showed better IC50 values in the peptide fractions with a molecular < 5 kDa weight in both legumes evaluated. Other researchers such as Chen et al.32 protein hydrolysates obtained from rice using the enzymes Alcalase and Trypsin and then these were fractionated by UF using two membranes of different molecular cut (3 to 10 kDa), and the values of IC50 were found in the fraction > 10 kDa = 1570 lg/mL, 3–10 kDa fraction = 840 lg/mL, and the fraction < 3 kDa = 280 lg/mL; the latter value is comparable with the results obtained in this study as fraction D obtained in 1– 3 kDa of P. lunatus hydrolyzed with Alcalase presented in IC50 = 262.5 lg/mL, which had better activity. These results are consistent with other reports of other investigators since ACE-I inhibitory activity increases as the molecular weight
6
BETANCUR-ANCONA ET AL.
of the peptide fractions decreases.12 Likewise, these differences in IC50 values could be due to the amino acid composition and sequence of the same; since each fraction showed that IC50 values were different, this feature will depend on the aforementioned factors. It is noteworthy that these amino acids have been previously found in other lupin protein hydrolysates,38 buckwheat,39 and azufrado beans (Phaseolus vulgaris).40 Furthermore, the presence of hydrophobic amino acids in peptide fractions of legumes evaluated showed high in V (54.3–82.5 g kg - 1), I (38.9–64.8 g kg - 1), P (4.3–101.8 g kg - 1), Met (18.7–27.4 g kg - 1), F (74.7–84.8 g kg - 1), L (102.9– 111.8 g kg - 1), and W (11.1–18.8 g kg - 1), and these amino acids are found in the amino acid sequence of different peptide fractions obtained from protein hydrolysates, as ‘‘GPP’’ for buckwheat,41 ‘‘LAIPVNSP’’ for soya,42 ‘‘VPP’’ for milk,43 ‘‘IFVPAF’’ for sea shrimp,44 and ‘‘IGDEPLANYL’’ for jellyfish.45 The ACE-I inhibitory activities imply that the hydrophobic amino acids may be present within the amino acid sequence of peptide fractions ( < 1 kDa) obtained from hydrolysates and P. vulgaris and P. lunatus with enzymes Alcalase and Flavourzyme, as they showed IC50 values of 30.3, 51.8, 63.8, and 65.8 lg/mL, respectively, on the ACE-I inhibition. Other researchers such as Ohba et al.46 reported P amino acid residues within the amino acid sequence of peptides found in the peptide fractions obtained ( < 1 kDa) from hydrolyzed collagen or keratin livestock and fish waste (bone and flesh), and these showed ACE-I inhibitory activity with an IC50 between 600 and 2800 lg/mL. Jung et al.37 found in the protein hydrolysate from skeleton Limanda aspera, the presence of hydrophobic amino acid residues within the amino acid sequence of a peptide, MIFPGAGGPEL, with a molecular weight of 1.3 kDa, and it showed ACE-I inhibitory activity with an IC50 = 28.7 lg/mL, which was similar to the values found in this study. Therefore, it has been recognized that the amino acid composition of the ACE-I inhibitory peptides is critical to possess inhibitory activity. Similarly, the structure–activity relationship indicates that the bond with ACE-I can be strongly affected by the tripeptide sequence in the Cterminal of the substrate and it is proposed that peptides include hydrophobic amino acids at these positions that are potent inhibitors of ACE-I.47 Therefore, our results suggest that ACE-I-inhibiting peptides found in peptide fractions < 1 kDa P. vulgaris and P. lunatus may contain hydrophobic amino acid residues in the C-terminal residues, especially P and A. Therefore, these results demonstrate to be important the structural identification of ACE-I inhibitory peptides in fractions of P. vulgaris and P. Iunatus. Since the primary activity of ACE-I is to cleave the Cterminal dipeptide of oligopeptide substrates with a wide specificity, the inhibitory activity of ACE-I inhibitory peptides is strongly influenced by their C-terminal tripeptide sequence. The most potent ACE-I inhibitors contain hydrophobic amino acid residues at each of the three Cterminal positions that interact with the subsites S1, S19, and S29 at the ACE-I active site.
Many studies have shown that peptides with high ACE-I inhibitory activities have tryptophan, phenylalanine, tyrosine, or proline at their C-terminus and branched aliphatic amino acids at the N-terminus. According to Cheung et al.,48 ACE-I is known to have little affinity for substrates or competitive inhibitor mechanisms with COOH-terminal dicarboxylic amino acids or with penultimate proline residues, but an antepenultimate aromatic amino acid residue appears to enhance binding. Bradykinin (Arg-Pro-Gly-PheSer-Pro-Phe-Arg) is bound more tightly to the enzyme than angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) and the pentapeptide substrate inhibitor; BPP is bound more tightly than either of these. In conclusion, protein hydrolysates, P. lunatus and P. vulgaris, obtained by enzymatic hydrolysis with Alcalase and Flavourzyme generated ACE-I inhibitory activity. Fractionation of these hydrolysates by UF originated peptide fractions of low-molecular weight ( < 1 kDa) that had a greater activity. Therefore, these fractions may have the commercial potential to be used as a functional ingredient for the prevention and/or treatment of hypertension.
ACKNOWLEDGMENTS This research was partially funded by the Consejo Nacional de Ciencia y Tecnologı´a (CONACYT) through doctoral scholarship 43867 and Project Ciencia Ba´sica 153012, the SIP-IPN (projects: 20060445, 20070800, and 20082532), and a scholarship from the Programa Institucional de Formacio´n de Investigadores (PIFI). AUTHOR DISCLOSURE STATEMENT No competing financial interests exist. REFERENCES 1. Chen YH, Liu YH, Yang YH, Feng HH, Chang CT, Chen CC: Antihypertensive effect of an enzymatic hydrolysate of chicken essence residues. Food Sci Technol Res 2002;8:144–147. 2. Scow TD, Smith GE, Shaughnessy FA: Combination therapy with ACE inhibitors and angiotensin-receptor blockers in heart failure. Clin Pharmacol 2003;68:1795–1798. 3. Hatanaka A, Miyahara H, Suzuki KI, Sato S: Isolation and identification of antihypertensive peptides from Antarctic krill tail meat hydrolysate. J Food Sci 2009;74:H116–H120. 4. Chiang WD, Tsou MJ, Tsai ZY, Tsai TC: Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor. Food Chem 2006; 98:725–732. 5. Copper OW, Herna´ndez-Diaz S, Arbogast GP, et al.: Major congenital malformations after first-trimester exposure to ACE inhibitors. N Engl J Med 2006;34:2443–2451. 6. Luna-Vital DA, Mojica L, Gonza´lez de Mejı´a E, Mendoza S, Loarca-Pin˜a G: Biological potential of protein hydrolysates and peptides from common bean (Phaseolus vulgaris L.): A review. Food Res Int [Epub ahead of print]; DOI: 10.1016/j.foodres .2014.11.024. 7. Cao W, Zhang C, Hong P, Ji H, Hao J: Purification and identification of an ACE inhibitory peptide from the peptic hydroly-
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