Purification of angiotensin I-converting enzyme inhibitory peptides from a cowpea (Vigna unguiculata) enzymatic hydrolysate

Process Biochemistry 46 (2011) 864–872

Contents lists available at ScienceDirect

Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Purification of angiotensin I-converting enzyme inhibitory peptides from a cowpea (Vigna unguiculata) enzymatic hydrolysate Maira Rubi Segura-Campos, Luis Antonio Chel-Guerrero, David Abram Betancur-Ancona ∗ Facultad de Ingeniería Química, Universidad Autónoma de Yucatán, Periférico Nte. Km. 33.5, Tablaje Catastral 13615, Col. Chuburná de Hidalgo Inn, 97203 Mérida, Yucatán, Mexico

a r t i c l e

i n f o

Article history: Received 29 October 2010 Received in revised form 13 December 2010 Accepted 15 December 2010 Keywords: Vigna unguiculata L. walp Hydrolysis Purification ACE inhibitors Amino acid composition

a b s t r a c t Angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated and characterized from cowpea (Vigna unguiculata L. walp). Cowpea protein isolate was prepared by wet fractionation, extensively hydrolyzed with Flavourzyme® and filtered through four molecular weight cut-off (MWCO) membranes in a high performance ultrafiltration (UF) cell. The ultrafiltered fraction with the highest ACE inhibitory activity was purified using gel filtration chromatography followed by reverse-phase high performance liquid chromatography (RP-HPLC). The cowpea peptide inhibition (IC50 ) values were similar to those reported for many other natural ACE inhibitory peptides. The <1 kDa ultrafiltered fraction exhibited the highest biological activity. The observed amino acid composition suggests a substantial contribution of hydrophobic residues to the peptides’ inhibitory potency, which potentially acts via blocking of angiotensin II production. When hydrolyzed with the protease Flavourzyme® , cowpea V. unguiculata protein is a good source of ACE inhibitory peptides with potential applications in physiologically functional foods with antihypertensive activity. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Angiotensin I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.1) is a vital component in blood pressure regulation. In the kinin–kallikrein system, ACE inactivates the vasodilator bradykinin, while in the renin–angiotensin system, ACE acts on the decapeptide angiotensin I to hydrolyze His-Leu from its C-terminal and produces the potent vasopressor octapeptide angiotensin II [1]. Angiotensin II is involved in release of the sodium-retaining steroid aldosterone, which increases blood pressure. ACE activity therefore increases blood pressure by raising vascular resistance and fluid volume. High blood pressure is associated with a higher risk of cardiovascular disease and stroke [2]. Among other applications, ACE inhibitors are used in prevention of hypertension, although their use in treatment of chronic heart failure and myocardial infarction has also been studied. The synthetic drugs currently used for hypertension treatment (e.g., Captopril® and Enalapril® ) have undesirable side effects, such as cough, skin discomfort and excessively low blood pressure [3]. In an effort to develop more effective ways of controlling blood pressure, both food and medical researchers have begun to investigate natural ACE inhibitors, such as bioactive peptides [4]. The first reported isolation of ACE inhibitory peptides from food proteins through the

∗ Corresponding author. Tel.: +52 9999460956; fax: +52 9999460994. E-mail address: [email protected] (D.A. Betancur-Ancona). 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.12.008

action of digestive proteases was performed over thirty years ago, and since then, many other ACE inhibitory peptides have been discovered in foods and by enzymatic digestion of food proteins [5], such as apricot [6], sardine [7], and milk [8]. Cowpea (Vigna unguiculata L. walp) is a major legume crop worldwide, particularly in tropical and subtropical areas, such as southeast Mexico. It serves as a major dietary protein source in both human and animal diets, and its protein content makes it a good raw material for preparation of protein isolates and hydrolysates, both of which are potential bioactive peptide sources [9]. The type of enzyme used in bioactive peptide preparations determines hydrolysate characteristics and the peptides that can be isolated. Flavourzyme® is a broad specificity fungal protease complex with exopeptidase activity that produces degree of hydrolysis (DH) values as high as 50% and results in hydrolysates containing mostly dipeptides [10]. Extensive hydrolysis of cowpea V. unguiculata protein isolate with Flavourzyme® could therefore produce a number of peptides with a myriad of potential applications; for example, as natural-source therapeutic elements in medical treatments and/or as an ingredient in functional foods. Ultrafiltration using a membrane system with different cutoff values is potentially useful in large-scale fractionation and enrichment of ACE inhibitory peptides [11]. Chromatography is a powerful technique for isolation and purification of bioactive peptides, and among the many chromatography techniques, high resolution fractionation by size exclusion chromatography and high-performance liquid chromatography (HPLC) are particularly

M.R. Segura-Campos et al. / Process Biochemistry 46 (2011) 864–872

865

Protein Hydrolysate

Soluble Fraction Ultrafiltration 10 K (MWCO) (>10 kDa) Retentate

Permeate (<10 kDa) 5 K (MWCO)

(5-10 kDa) Retentate

Permeate (<5 kDa) 3 K (MWKO) (MWCO)

(3-5 kDa) Retentate

Permeate (<3 kDa) 1 K (MWCO)

Permeate (<1 kDa)

(1-3 kDa) Retentate

Fig. 1. Process flow diagram of V. unguiculata fractionation [15].

well-suited to function as the final step in a purification regimen [12]. The present study objective was to use gel filtration chromatography and HPLC to purify ACE inhibitory peptides from cowpea V. unguiculata protein hydrolysates filtered with a high performance ultrafiltration cell, and quantify the resulting peptides’ amino acid composition. Considering the results, the potential of cowpea as an ingredient for the production of antihypertensive functional foods is discussed. 2. Materials and methods

4% (w/v) protein solution, and the solution was equilibrated at optimum temperature (50 ◦ C) and pH (7). Flavourzyme® was then added to the solution at a ratio of 50 LAPU/g protein, and pH was kept constant by adding 1.0 M NaOH during hydrolysis. The reaction was stopped by heating to 80 ◦ C for 20 min, followed by centrifuging at 9880 × g for 20 min to remove the insoluble portion. 2.4. Degree of hydrolysis DH was calculated by calculating free amino groups with o-phthaldialdehyde [14]: DH = h/htot × 100, where htot is the total number of peptide bonds per protein equivalent, and h is the number of hydrolyzed bonds. The htot factor is dependent on raw material amino acid composition.

2.1. Materials

2.5. Hydrolysate fractionation by ultrafiltration

Cowpea V. unguiculata L. walp seeds were obtained from the February 2007 harvest in the state of Yucatan, 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). Flavourzyme® 500MG enzyme was purchased from Novo Laboratories (Copenhagen, Denmark).

Following Cho et al. [15], the hydrolysate was fractionated by ultrafiltration using a high performance ultrafiltration cell (Model 2000, Millipore, Inc., Marlborough, MA). Five fractions were prepared using four molecular weight cut-off membranes: 1 kDa, 3 kDa, 5 kDa and 10 kDa. Soluble fractions prepared by centrifuging were passed through the membranes stating with the largest MWCO membrane cartridge (10 kDa). The retentate and permeate were collected separately, and the retentate recirculated into the feed until maximum permeate yield was reached, as indicated by a decreased permeate flow rate. The permeate from the 10 kDa membrane was then filtered through the 5 kDa membrane with recirculation until maximum permeate yield was reached. The 5 kDa permeate was then processed with the 3 kDa membrane and the 3 kDa permeate with the 1 kDa membrane (Fig. 1). This process minimized contamination of the larger molecular weight fractions with smaller molecular weight fractions while producing enough retentates and permeates for the following analyses. The five ultrafiltered peptide fractions were designated as >10 kDa (10 kDa retentate); 5–10 kDa (10 kDa permeate–5 kDa retentate); 3–5 kDa (5 kDa permeate–3 kDa retentate); 1–3 kDa (3 kDa permeate–1 kDa retentate); and <1 kDa (1 kDa permeate).

2.2. Protein isolates A single extraction was performed with 6 kg of cowpea seeds. Impurities and damaged seeds were removed, and sound seeds milled in a Mykros impact mill until passing through a 20-mesh screen (0.85 mm) followed by milling in a Cyclotec 1093 (Tecator, Sweden) mill until passing through a 60-mesh screen (0.24 mm). The resulting flour was processed using the wet fractionation method of BetancurAncona et al. [13]. Briefly, whole flour was suspended in distilled water at a 1:6 (w/v) ratio, pH was adjusted to 11 with 1 M NaOH, and the dispersion was stirred for 1 h at 400 rpm with a mechanical agitator (Caframo Rz-1, Heidolph Schwabach, Germany). This suspension was wet-milled with a Kitchen-Aid® food processor, and the fiber solids were separated from the starch and protein mix by straining through 80and 150-mesh sieves and washing the residue five times with distilled water. The protein–starch suspension was allowed to sediment for 30 min at room temperature to recover the starch and protein fractions. The pH of the separated solubilized proteins was adjusted to the isoelectric point (4.5) with 1 N HCl. The suspension was then centrifuged at 1317 × g for 12 min (Mistral 3000i, Curtin Matheson Sci.), the supernatants were discarded, and the precipitates were freeze-dried at −47 ◦ C and 13 × 10−3 mbar until use. 2.3. Enzymatic Hydrolysis The protein isolates were hydrolyzed with Flavourzyme® 500MG (Novo Nordisk, Bagsvaerd, Denmark) for 90 min under controlled conditions (temperature, pH and stirring) in a 1000 mL reaction vessel equipped with a stirrer, thermometer and pH electrode [8]. Protein isolates were suspended in distilled water to produce a

2.6. ACE inhibitory activity ACE inhibitory activity in the hydrolysate and its purified peptide fractions was analyzed following Hayakari et al. [16]. ACE hydrolyzes hippuryl-l-histidyll-leucine (HHL) to yield hippuric acid and histidyl-leucine. This method relies on the colorimetric reaction of hippuric acid with 2,4,6-trichloro-s-triazine (TT) in a 0.5 mL incubation mixture containing 40 ␮mol potassium phosphate buffer (pH 8.3), 300 ␮mol sodium chloride, 40 ␮mol 3% HHL in potassium phosphate buffer (pH 8.3) and 100 mU/mL ACE. The mixture was incubated at 37 ◦ C for 45 min and then reaction terminated by addition of TT (3%, v/v) in dioxane and 3 mL of 0.2 M potassium phosphate buffer (pH 8.3). After centrifuging the reaction mixture at 10,000 × g for 10 min, enzymatic activity was determined in the supernatant by measuring absorbance at 382 nm. All runs were performed in triplicate. ACE inhibitory activity was quantified by a regression analysis of ACE inhibitory activity (%) versus peptide concentration and defined as an IC50 value, that is, the peptide concentration

866

M.R. Segura-Campos et al. / Process Biochemistry 46 (2011) 864–872

in (␮g protein/mL) required to produce 50% ACE inhibition under the described conditions. 2.7. G-50 gel filtration chromatography After filtration through 10, 5, 3 and 1 kDa membranes in a high performance ultrafiltration cell, 10 mL of the fraction with highest ACE inhibitory activity was injected into a Sephadex G-50 gel filtration column (3 cm × 79 cm) at a flow rate of 25 mL/h of 50 mM ammonium bicarbonate (pH 9.1). The resulting fractions were collected for to assay ACE inhibitory activity according to Hayakari et al. [16]. Peptide molecular masses were determined by reference to a calibration curve created by running molecular mass markers on the Sephadex G-50 under conditions identical to those used for the test samples. Molecular mass standards were thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), equine myoglobin (17 kDa), vitamin B12 (1.35 kDa) and Thr-Gln (0.25 kDa). Fractions selected for further purification of peptides were pooled and lyophilized before RP-HPLC. 2.8. HPLC C18 chromatography The fractions isolated with the Sephadex G-50 column were redissolved in deionized water and injected into a preparative HPLC (Agilent, Model 1110) reverse-phase column (C18 Hi-Pore RP-318, 250 mm × 10 mm BIO-RAD column). The injection volume was 100 ␮L, and the sample concentration was 20 mg/mL. Elution was achieved by a linear gradient of acetonitrile in water (0–30% in 50 min) containing 0.1% trifluoroacetic acid at a flow rate of 4 mL/min and 30 ◦ C [17]. Elution was monitored at 215 nm, and the resulting fractions were collected for assay of ACE inhibitory activity as described above [16]. 2.9. Amino acid composition Protein amino acid composition was determined for the hydrolysate, and the peptides were purified by ultrafiltration, gel filtration chromatography and HPLC [18]. Samples (2–4 mg protein) were treated with 4 mL of 6 mol equi L−1 HCl, placed in hydrolysis tubes and gassed with nitrogen at 110 ◦ C for 24 h. They were then dried in a rotavapor and suspended in 1 mol L−1 sodium borate buffer at pH 9.0. Amino acid derivatization was performed at 50 ◦ C using diethyl ethoxymethylenemalonate. Amino acids were separated using HPLC with a reversed-phase column (300 × 3.9 mm, Nova Pack C18, 4 mm; Waters), and a binary gradient system with 25 mmol L−1 sodium acetate containing (A) 0.02 g L−1 sodium azide at pH 6.0, and (B) acetonitrile as solvent. The flow-rate was 0.9 mL min−1 , and the elution gradient was: 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). 2.10. Statistical analysis All results were analyzed in triplicate using descriptive statistics with a central tendency and dispersion measures. One-way ANOVAs were performed to evaluate protein isolate hydrolysis data and in vitro ACE inhibitory activity. A Duncan’s multiple range test was used to determine differences between treatments. All analyses were performed according to Montgomery [19] and processed using the Statgraphics Plus version 5.1 software.

3. Results and discussion 3.1. Protein isolate hydrolysis Hydrolysis with Flavourzyme® produced an extensively hydrolyzed V. unguiculata protein hydrolysate. Enzymatic hydrolysis is widely used to produce food-grade protein hydrolysates and to release bioactive peptides from their protein precursors [12]. In the present study, hydrolysis of cowpea proteins with Flavourzyme® resulted in a maximum DH of 58.8% at an enzyme concentration of 50 leucine amino peptidase units (LAPU)/g protein, a 90 min hydrolysis time and stable pH conditions (pHstat). This DH is higher than reported for defibrinated bovine plasma hydrolysates (48.3% DH) produced with Flavourzyme® at 100 LAPU/g protein for 24 h [20] and higher than chickpea protein hydrolysates (27% DH) produced by treatment with Flavourzyme® for 180 min [21]. The very high DH recorded for the cowpea hydrolysate is the result of Flavourzyme’s® broad specificity, which produces DHs as high as 50% and a hydrolysate containing mainly dipeptides. However, the hydrolysis conditions used in the present study, such as close control of pH, were also at least partially

responsible for this high DH. Different raw materials produce varying DHs that are dependent on hydrolysis conditions such as pH. For instance, Flavourzyme® hydrolysis of a cowpea V. unguiculata protein concentrate in a system with uncontrolled pH produced a hydrolysate with only 7.27% DH after 60 min [22]. Imm and Lee [23] reported that dissociation of the ␣-amino group increases when hydrolysis is performed at pH >6.5 and that the resulting increase in the number of protonated ␣-amino groups by cleavage of peptide bonds causes a decreases in pH because the carboxyl groups are not readily protonated at this pH range. When hydrolyzing a soy protein isolate using a fungal origin enzyme complex such as Flavourzyme® , Segura et al. [10] found that hydrolysis was more efficient under natural, drifting pH than controlled pH conditions. Similarly, Wanasundara et al. [20] reported that when hydrolyzing defibrinated bovine plasma, Flavourzyme® exhibited optimum activity (48.3% DH) under conditions of natural pH drift between 7.0 and 5.0, rather than stable pH (pH-stat) conditions. In contrast, in a study of hydrolysis of mullet (Mugil cephalus) using a bacterial protease, Rebeca et al. [24] reported that protein hydrolysis was more rapid under controlled pH conditions during the first 2 h, although no difference in soluble nitrogen content was observed between uncontrolled and controlled pH systems at 3 h of hydrolysis. The high DH observed in the present study suggests that Flavourzyme® hydrolysis of cowpea V. unguiculata protein isolate is most efficient under controlled pH conditions. 3.2. ACE inhibitory activity Hydrolysate ACE inhibitory activity, measured and calculated as IC50 , was 2634.4 ␮g/mL. The IC50 value was lower than reported for shrimp (3370 ␮g/mL) but higher than reported for Alaska pollak protein hydrolysates (210 ␮g/mL), blue mussel sauce (19.34 ␮g/mL), sardine (15 ␮g/mL) and sea bream (0.57 ␮g/mL) [7]. Inhibitory activity was also determined for the permeates and retentates produced by ultrafiltration of the hydrolysate in response to reports that small peptides make a considerable contribution to the ACE inhibitory activity of protein hydrolysates and that short peptide sequences are good candidates to be in vivo physiological antihypertensive agents [25]. Ultrafiltration of the hydrolysate produced peptide fractions with increased biological activity. Inhibitory activity (IC50 ) was dependent (p < 0.05) on peptide fraction molecular weight, the lowest activity being in the >10 kDa fraction and the highest in the <1 kDa fraction: 0.04 ␮g/mL (F < 1 kDa); 31.1 ␮g/mL (F1–3 kDa); 85.4 ␮g/mL (F3–5 kDa); 89.7 ␮g/mL (F5–10); and 170.6 ␮g/mL (F > 10 kDa). This coincides with other reports of increased ACE inhibitory activity as hydrolysate fraction molecular weight decreases. The present results also confirm that ultrafiltration membranes can be used to enrich specific peptide fractions. Je et al. [26] reported that the fraction having a molecular mass below 1 kDa from Alaska Pollack (Theragra chalcogramma) frame protein hydrolysate showed the highest ACE inhibitory activity (87.62%, IC50 = 457 ␮g/mL) than that F1–3 kDa (78.95%), F3–5 kDa (73.84%), F5–10 kDa (68.24%), and F10–30 kDa (59.60%) fractions. Korhonen and Pihlanto [27] reported that the <1 kDa fractions from ␣-lactalbumin hydrolyzed with pepsin, or ␤-lactalbumin hydrolyzed with pepsin–trypsin or pepsin–trypsin–chymotrypsin combinations, exhibited higher ACE inhibitory activity than other fractions. These results, combined with those of Segura et al. [10], suggest that ultrafiltration is a promising way of enriching ACE-inhibitory peptides derived from cowpea protein. Zhenbao et al. [6] confirmed the above mentioned results using ultrafiltered apricot kernel protein hydrolysates on membranes with different MWCO and to observe that most of the ACE inhibitory activity was due to peptides that were less than 1 kDa in molecular weight; the IC50 value of the 1 kDa ultrafiltrate was 150 ␮g/mL while it was 378 ␮g/mL before ultrafiltration.

M.R. Segura-Campos et al. / Process Biochemistry 46 (2011) 864–872

Fig. 2. Elution profile of the <1 kDa ultrafiltration fraction of the cowpea V. unguiculata protein hydrolysate purified in a Sephadex G-50 gel filtration column. superscripts letters indicate statistical difference (p < 0.05). Data are the mean of three replicates.

867

a–e

Different

This has been demonstrated with other raw materials. For instance, ultrafiltered thermolysin digest from dried bonito had more potent antihypertensive effects in hypertensive subjects than the source hydrolysate [28]. Mao et al. [4] concluded that the lower was the molecular weight of the yak casein hydrolysate fraction, the higher was its ACE inhibitory activity, and the molecular weight of the most effective fraction was below 6 kDa (85.4%).

and 1000 Da and IC50 values of 120 ␮g/mL and 88 ␮g/mL, respectively [31]. Ji-Eun et al. [32] reported similar results to separate of size exclusion chromatography peptide fractions below 3 kDa (IC50 = 500 ␮g/mL) from textured and fermented vegetable protein IC50 = 2190 ␮g/mL). They purified peptide fraction with a molecular weight range of 500–999 Da with IC50 values of 94 ␮g/mL and that represented peptides of approximately 7 amino acids residues.

3.3. Gel filtration chromatography of the <1 kDa fraction

3.4. Reverse-phase HPLC chromatography of pooled fractions

Because it exhibited the highest ACE inhibitory activity, the <1 kDa fraction was selected for further fractionation. Gel filtration chromatography (Sephadex G-50 column) was used to generate a molecular weight profile of the <1 kDa fraction. The profile was typical of a protein hydrolysate formed by a pool of peptides, with gradually decreasing molecular masses. Elution volumes between 406 and 518 mL included free amino acids and peptides with molecular masses ranging from 3.6 to 0.4 kDa. This range was fractionated into eleven fractions (1–11) and ACE inhibitory activity determined for each. Fractions with elution volumes smaller than 406 mL and greater than 518 mL were not analyzed because they largely included peptides with high molecular weights, as well as free amino acids. ACE inhibitory activity (%) in the eleven fractions ranged from 5.29 to 47.43% and differed (p < 0.05) between fractions. The highest ACE inhibitory activity was observed in fractions F4 (47.43%; 437.5–444.5 mL elution volume) and F5 (45.14%, 448–455 mL elution volume), which were not statistically different (p < 0.05) (Fig. 2). Their molecular masses were approximately 1.8 kDa (indicative of 7 amino acid residues) and 1.5 kDa (indicative of 10 amino acid residues), respectively. The IC50 value for F4 (14.19 ␮g/mL) was similar than those of Fagopirum esculentum peptide fractions purified by Sephadex LH-20 gel filtration (15.1 ␮g/mL) [5], but lower than those of gel filtration (Sephadex G-25) peptide fractions from tuna broth hydrolysate (210–25,260 ␮g/mL) [29], from Fagopyrum esculentum Moench (Sephadex C-25 = 25,715.1 ␮g/mL; Sephadex G-10 = 21,315.1 ␮g/mL), and from the peptic hydrolysate of Acetes chinensis (Sephadex C-15 = 770–1590 ␮g/mL) [30]. A similar behavior pattern was observed with Konjac peptides purified by series connection of Sephadex G-25 and Sephadex G-15 columns that resulted in purified peptides with molecular weights of 1500

Fractions F4 and F5 from the gel filtration chromatography treatment were pooled and analyzed using RP-HPLC to produce a chromatographic profile from mass-transfer between stationary and mobile phases (Fig. 3). Mixture components were separated by dissolving fractions F4 and F5 in acetonitrile and forcing them through a chromatographic column (C18 Hi-Pore RP-318, 250 mm × 10 mm BIO-RAD column) under high pressure. The mixture resolved into its components in the column, separating F4 and F5 based on differences in hydrophobicity. In this process, the components of both fractions passed over stationary-phase particles containing pores large enough for them to enter, and in which interactions with the hydrophobic surface removed them from the flowing mobile-phase stream. The strength and nature of the interaction between the sample particles and stationary phase depended on hydrophobic and polar interactions. As the eluent organic solvent concentration increased, it reached a critical value for each analyte and desorbed it from the hydrophobic stationaryphase surface to allowing it to elute from the column into the flowing mobile phase. Because this elution depended on the precise distribution of hydrophobic residues in each species, each analyte eluted from the column at a characteristic time and the resulting peaks were used to qualitatively analyze both fractions’ components. Within each gel filtration fraction, the eluates were divided into four major fractions: F4-1, F4-2, F4-3, F4-4; F5-1, F5-2, F5-3, F5-4 (Fig. 3). The peptides were relatively pure, although a small shoulder still appeared behind the peaks in the chromatogram. Enough material from each fraction was collected in successive analyses to determine ACE inhibitory activity (Fig. 4). Fraction F4 had a larger (p < 0.05) ACE inhibitory activity range (33.83–75.42%) than F5 (32.31–49.71%). Overall, F4-2 (75.42%) had the highest ACE inhibitory activity (IC50 = 0.4704 ␮g/mL). This value is lower

868

M.R. Segura-Campos et al. / Process Biochemistry 46 (2011) 864–872

3.5. Amino acid composition

Fig. 3. Preparative RP-HPLC C-18 chromatography of the bioactive fractions (a) F4 and (b) F5 purified by gel filtration.

than the 6.3 ␮g/mL reported for a peptide from F. esculentum Moench purified by RP-HPLC [5] and within the 8.1–91.6 ␮g/mL range reported for fractions from caprine kefir water-soluble extract purified by preparative RP-HPLC [33]. The same behavior was observed when comparing the results of RP-HPLC fractions obtained of Mustelus mustelus intestines with alkaline proteases (130–783 ␮g/mL) [1] and with peptide fractions obtained from the peptic hydrolysate of the freshwater rotifer Brachionus calyciflonus (40.01 ␮g/mL) [34].

Fig. 4. ACE inhibition percentage of RP-HPLC fractions. are the mean of three replicates.

a–c

Fractions with the highest ACE inhibitory activity were analyzed to produce an amino acid profile. During hydrolysis, asparagine and glutamine partially converted to aspartic acid and glutamic acid, respectively; the data for asparagine and/or aspartic acid were therefore reported as Asx while those for glutamine and/or glutamic acid were reported as Glx. The higher ACE inhibitory activity exhibited by the <1 kDa fraction (IC50 = 0.04 ␮g/mL) compared to the Flavourzyme hydrolysate (IC50 = 2634.4 ␮g/mL) was probably due to its higher concentration of neutral amino acids, such as Ser (3.03%) and Thr (11.36%), hydrophilics such as His (12.5%) or hydrophobics such as Ala (7.84%), Pro (23.80%), Val (10%), Met (66.66%), Ile (10%), Leu (21.6%), Phe (18.6%) and Trp (16.6%) (Table 1). Compared to the <1 kDa fraction amino acid profile, the G-50 gel filtration chromatography fractions had higher Asx, Glx and Arg concentrations. Hydrophobic amino acid content decreased by 20.27% in F4 (34.56 g/100 g) and 21.19% in F5 (34.16 g/100 g) compared to the <1 kDa fraction (43.35 g/100 g), while hydrophilic residues increased by 19.25% (48 g/100 g) in F4 and 22.48% (50 g/100 g) in F5. The ACE inhibitory activity observed in F4 (47.43%) and F5 (45.14%) could therefore be the result of their higher Arg, Asx or Asp concentrations. These residues are known to play an important role in the antihypertensive activity of peptides from white and red wines and from French flor-sherry wine [35]. The F4 and F5 fractions may also have the added benefit of low bitterness. Many of the ACE-inhibitory peptides isolated from food sources are composed of multiple food components and hydrophobic and/or aromatic amino acid residues. However, practical use of these food protein hydrolysates is complicated by formation of peptides which impart a bitter taste, the result of the formation of low molecular weight peptides containing mostly hydrophobic amino acids. To address this problem, Kim et al. [11] recommended use of Flavourzyme® , a fungal endoprotease and exo-protease complex that produces hydrolysates or peptides with ACE inhibitory activity and low bitterness. Fractions F4 and F5 are promising prospects for use in new product development because they had clear ACE inhibitory activity and low hydrophobic amino acid content, which may ensure that they have low bitterness. Comparison of amino acid composition and properties between the cowpea V. unguiculata hydrolysate and its peptides isolated by ultrafiltration and G-50 gel chromatography showed the most

Different letters in the same gel filtration chromatography fraction indicate statistical difference (p < 0.05). Data

M.R. Segura-Campos et al. / Process Biochemistry 46 (2011) 864–872

869

Table 1 Amino acid contents of the cowpea V. unguiculata protein concentrate (PC), Flavourzyme hydrolysate (FH), <1 kDa ultrafiltered fraction and F4 and F5 gel filtration chromatography fractions. Amino acid

Composition (g/100 g)a PC

Asx Glx Ser His Gly Thr Arg Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Trp a

10.8 19 6.5 2.9 4.4 4.3 7.8 4.4 2.7 2.6 5.4 0.1 0.4 4.4 9.2 6.9 7.3 0.7

FH ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.003 0.016 0.041 0.028 0.005 0.009 0.067 0.008 0.078 0.098 0.052 0.109 0.002 0.019 0.115 0.008 0.089 0.064

9.6 18.6 6.4 2.8 4.5 3.9 7.9 4.7 1.6 2.7 6.3 0.3 0.4 5.4 9.8 7.0 7.1 1.0

F < 1 kDa ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.387 0.013 0.016 0.001 0.006 0.009 0.025 0.002 0.038 0.006 0.015 0.001 0.006 0.207 0.175 0.004 0 0.038

9.4 14.9 6.6 3.2 4.1 4.4 6.0 5.1 2.1 2.6 7.0 0.9 0.2 6.0 12.5 8.6 5.3 1.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.448 0.171 0.046 0.024 0.035 0.032 0.073 0.106 0.01 0.026 0.029 0.013 0.002 0.073 0.029 0.056 0.072 0.041

F4 14 17.9 5.8 2.9 4.3 4.0 7.5 4.3 0 2.2 5.3 0.7 1.2 5.2 11.6 6.7 5.7 0.8

F5 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.274 0.182 0.302 0.008 0.011 0.040 0.026 0.016 0 0.018 0.004 0.006 0.01 0.015 0.092 0.046 0.042 0.241

16.7 18.3 5.0 2.7 3.0 3.4 6.9 3.6 0 1.2 4.2 2.2 3.2 4.7 12.6 5.8 5.3 1.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.152 0.17 0.126 0.061 0.177 0.036 0.039 0.625 0 0.078 0.211 0.292 0.1 0.236 0.229 0.043 0.263 0.535

Data are the mean of three replicates.

active fraction to be the <1 kDa fraction, which had abundant aromatic (e.g., Phe) and cyclic amino acids (e.g., Pro) (Fig. 5). Amino acids such as Phe, with large bulky chains and hydrophobic side chains, are preferred in both positions of a dipeptide for their high steric properties and low lipophilicity, while amino acids such as Pro are preferred in the carboxyl terminus of active tripeptides for their low lipophilicity, and high steric and electronic properties [36]. Aromatic and basic amino acids are important to the ACE inhibitory activity of peptides. For instance, similar observations have been made for an orientase hydrolysate with notable ACE inhibition attributed to its high basic and aromatic amino acids contents [30]. Cheung et al. [37] reported a peptide with strong, competitive ACE inhibition in which aromatic amino acid residues at its C-terminal and basic or hydrophobic ones at its N-terminal played an essential role. Based on the above, the higher ACE inhibitory potential of the <1 kDa fraction from the cowpea hydrolysate can be attributed to the steric properties of its aromatic amino acids and the lipophilicity and electronic properties of its cyclic amino acids. The most active fraction among those purified from F4 by preparative RP-HPLC was F4-2, which had a much higher neu-

tral amino acid content (80.6 g/100 g) than F4-1 (46.6 g/100 g), F4-3 (6.1 g/100 g) and F4-4 (28.6 g/100 g) (Table 2). Of the neutral amino acids, Tyr was higher in F4-2 (71.3 g/100 g) compared to F4-3 (1.5 g/100 g), F4-4 (3.9 g/100 g) and F4-1 (3.9 g/100 g). This supports the importance of aromatic residues in a peptides’ biological potential, probably due to their high steric properties and low lipophilicity (Fig. 6a). The Tyr amino acid has been reported in peptides from milk (e.g., Tyr-Pro-Tyr-Tyr) isolated by a combination of lactic acid bacteria fermentation and Flavourzyme® hydrolysis. These peptides are bioavailable and exhibit in vitro (90.9 ␮M) and in vivo ACE inhibitory activity, the latter in the form of reduced hypertension in spontaneously hypertensive rats (SHR) (15.9 mm Hg reduction in SBP) [8]. However, F4-2 was also unique in containing Met (0.5 g/100 g) and Trp (0.2 g/100 g), as well as higher concentrations of Ile (3.2 g/100 g) and Leu (10.2 g/100 g) than the other fractions eluted from F4, all of which could have significantly increased its relative ACE inhibition activity. This would coincide with previous reports of inhibitory activity in peptides with these amino acids. The residues Val, Leu and Ile are preferred in the amino terminal position in active tripeptides for their low lipophilicity and steric properties or side chain bulk/molecular size [36].

Fig. 5. Amino acid distribution in the cowpea V. unguiculata protein concentrate, Flavourzyme hydrolysate, <1 kDa ultrafiltered fraction, and F4 and F5 gel filtration chromatography fractions. N: neutral amino acids (including Gly, Ala, Ser, Thr, Val, Leu and Ile). A: acid amino acids (including Asp and Glu). B: basic amino acids (including Lys, His and Arg). Ar: aromatic amino acids (including Phe, Tyr and Trp). C: cyclic amino acids (including Pro).

870

M.R. Segura-Campos et al. / Process Biochemistry 46 (2011) 864–872

Fig. 6. Amino acid distribution of fractions F4 (a) and F5 (b), purified by preparative RP-HPLC C-18 chromatography. N: neutral amino acids (including Gly, Ala, Ser, Thr, Val, Leu and Ile). A: acid amino acids (including Asp and Glu). B: basic amino acids (including Lys, His and Arg). Ar: aromatic amino acids (including Phe, Tyr and Trp). C: cyclic amino acids (including Pro).

Clearly, the structure of ACE inhibitory peptides influences their activity, as shown by Cheung et al. [37], who reported that peptides with Tyr at the C-terminus and Ile at the N-terminus exhibit highly potent inhibitory activity. In a randomized, double-blind, placebo-controlled human study, a significant depressor effect was Table 2 Amino acid contents of fractions from the F4 gel filtration chromatography fraction purified by preparative RP-HPLC C-18 chromatography. Amino acid

Asx Glx Ser His Gly Thr Arg Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Trp a

Content (g/100 g)a F4-1

F4-2

9.9 ± 0.063 5.1 ± 0.214 22.7 ± 0.291 8.2 ± 0.677 19.6 ± 0.983 4.3 ± 0.987 3.2 ± 0.065 14.3 ± 0.620 0±0 0±0 0±0 0±0 0±0 0.4 ± 0.555 9.3 ± 0.217 0±0 3 ± 0.532 0±0

1.2 1.4 1 0.4 1.4 0.3 0.8 0.4 0 71.3 0 0.5 6.6 3.2 10.2 0.8 0.3 0.2

F4-3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Data are the mean of three replicates.

0.009 0.048 0.001 0.015 0.008 0.005 0.002 0.001 0 0.012 0 0.013 0.001 0.041 0.006 0.003 0.007 0.040

0.8 1.1 1.2 0.3 1.7 0.4 0.8 0.3 0 1.5 0 0 1.3 0.2 0.3 89.6 0.3 0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

F4-4 0.001 0.006 0.021 0.009 0.031 0.018 0.01 0.005 0 0.026 0 0 0.085 0.020 0.008 0.230 0.008 0

12.5 12.6 8.4 4.1 9.2 3.5 7.1 3.8 0 3.9 0 0 3.6 2.8 4.7 20 3.8 0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.242 0.329 0.177 0.133 0.226 0.066 0.151 0.046 0 0.109 0 0 0.288 0.059 0.024 0.778 0.068 0

observed in mild essential hypertensive volunteers [38] and Val-Tyr was shown to be one of the predominant ACE inhibitory peptides involved in this effect [39]. In addition to its high Tyr content, F4-2 may also be absorbed intact into the human circulatory system and induce a reduction in blood pressure. For instance, intravenous and oral administration studies of Val-Tyr in SHR have shown that the small dipeptide Val-Tyr caused a long-lasting depressor effect [40]. Val-Tyr is known to be absorbed intact into the human circulatory system [41], and studies using cross-mated transgenic mice carrying the human renin gene and the human angiotensinogen gene have shown that, as a natural ACE inhibitory dipeptide, Val-Tyr regulates the enhanced human renin–angiotensin system and induces a prolonged reduction in blood pressure [42]. Of the fractions purified from F5 by preparative RP-HPLC chromatography (Table 3), F5-2, F5-3 and F5-4 exhibited ACE inhibition that was not different among them (p < 0.05), but was greater than that of F5-1. The amino acids Arg, Tyr, Met, Ile, Leu, Phe and Lys very probably played a key role in providing greater activity to the first three fractions. As mentioned above, residues such as Leu and Ile are preferred at the amino terminus of tripeptides with ACE inhibitory activity due to their low lipophilicity and steric properties or side chain bulk/molecular size values. In addition, Lys and Arg are expected in positions adjacent to the amino terminus due to their low electronic properties and high lipophilicity and steric property values, while residues such as Phe are preferred at the carboxyl terminus due to their low lipophilicity and high steric and electronic property values [36]. Although the precise substrate specificity is not fully understood, ACE appears to pre-

M.R. Segura-Campos et al. / Process Biochemistry 46 (2011) 864–872 Table 3 Amino acid contents of fractions from the F5 gel filtration chromatography fraction purified by preparative RP-HPLC C-18 chromatography. Amino acid

Content (g/100 g)a F5-1

Asx Glx Ser His Gly Thr Arg Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Trp a

2.1 0 30.5 16.3 13.3 5.8 0 1.1 0 0 0 0 31 0 0 0 0 0

F5-2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.492 0 0.834 0.194 0.159 0.069 0 1.536 0 0 0 0 0.369 0 0 0 0 0

1.2 1.5 0.9 0.3 2.9 0.3 0.2 0.3 0 74.7 0 0.6 8.8 1.6 3.1 3.5 0.2 0

F5-3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.005 0.046 0.014 0.049 0.017 0.048 0.236 0.048 0 0.280 0 0.071 0.098 0.035 0.066 0.765 0.009 0

2 2 1 0.4 2 0.4 0.6 0.5 0 0.7 0 0.1 0.9 0.5 0.8 87.9 0.3 0

F5-4 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.004 0.071 0.014 0.017 0.016 0.024 0.028 0.012 0 0.109 0 0.096 0.019 0.026 0.093 0.234 0.002 0

10.6 14.2 8.4 2.7 10 2.7 3 3.4 0 4.2 0 0.5 6.8 1.8 6.1 22.4 2.4 0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.731 0.762 0.009 0.957 0.057 0.957 0.139 0.089 0 0.289 0 0.039 0.070 0.032 0.896 0.304 0.073 0

Data are the mean of three replicates.

fer substrates containing hydrophobic amino acid residues at the three C-terminal positions, suggesting that the higher hydrophobic amino acid content in F5-2 (9.1 g/100 g), F5-3 (89.8 g/100 g) and F5-4 (34.2 g/100 g) versus F5-1 (1.1 g/100 g) probably made a substantial contribution to their inhibitory potency (Fig. 6b). This agrees with Wu et al. [36], who state that aromatic, positively charged and hydrophobic amino acids are preferred in active tripeptides. Due to substrate specificity differences between the two ACE catalytic sites, ACE inhibitors may inhibit only one site. Moskowitz [43] proposed a model explaining the clinical superiority of hydrophobic ACE inhibitory drugs relative to hydrophilic ones: all ACE inhibitors bind to the C-terminal catalytic site, but only hydrophobic ones bind to the occluded N-terminal catalytic site and are therefore better at blocking angiotensin II production. This would also explain why hydrophobic ACE inhibitors have specific local benefits such as organ damage prevention, in addition to reducing blood pressure [43]. The high hydrophobic amino acid (particularly aromatic side-chains) content in the F5 may therefore make a substantial contribution to its fractions’ ACE inhibitory activity by blocking angiotensin II production. Overall, the highest in vitro ACE inhibitory activity (IC50 ) among the cowpea hydrolysate and its derivative fractions was present in the ultrafiltered <1 kDa fraction (0.04 ␮g/mL), followed by the RP-HPLC F4-2 fraction (0.4704 ␮g/mL), the gel filtration chromatography fraction F4 (14.195 ␮g/mL) and finally the hydrolysate (2634.4 ␮g/mL) (Table 4). Although the IC50 values for the hydrolysate, F4 and F4-2 fractions were substantially higher than that of the F < 1 kDa fraction, they are still in the same order of magnitude as values reported for many other natural ACE inhibitory peptides. Nevertheless, the IC50 values for all the studied V. unguiculata derivatives are far higher than that of the synthetic ACE inhibitor Captopril® (0.0013 ␮g/mL) [29]. The biological potential in the peptides purified from V. unguiculata, and the high ACE

Table 4 Comparative ACE inhibitory activity (IC50 ) of the cowpea V. unguiculata derivatives by purification step. Purification step

IC50 (␮g protein/mL)

FH UF (F < 1 kDa) Sephadex G-50 (F4) RP-HPLC (F4-2)

2634.4 0.04 14.195 0.4704

871

inhibitory activity in the F < 1 kDa fraction, reinforce the need for ACE inhibitory peptides to be rich in hydrophobic amino acids (aromatic or branched chains) [44] and peptides rich in proline [45]. Proline is well-documented as the most favorable amino acid for ACE binding (most commercial inhibitors include this residue), but it was not present in the peptides with potential biological activity that had been purified by G-50 gel filtration chromatography and RP-HPLC. The studied cowpea V. unguiculata protein hydrolysate has potential applications in the development of physiologically functional foods aimed at preventing and/or treating hypertension. An added benefit is the balanced amino acid profile of the protein hydrolysate and its peptide fractions, which makes them an appropriate protein source in human nutrition. It should be considered, however, that the in vitro ACE inhibitory potencies of peptides do not always correlate with their in vivo antihypertensive activities as quantified in SHR [46]. This is because they must be absorbed and transported intact from the intestine to the blood stream (in the case of oral administration) and resist plasma peptidase degradation (in the case of oral and intravenous administration) to reach their target sites and exert an antihypertensive effect in vivo. Therefore, in vivo research is needed to determine to what extent any of the studied ACE inhibitory peptides can exercise their antihypertensive activity in vivo. 4. Conclusions After modification by the food-grade microbial enzyme Flavourzyme, cowpea V. unguiculata proteins proved to be a source of bioactive peptides with ACE inhibitory activity. Further purification of the peptides by ultrafiltration, gel filtration chromatography and RP-HPLC produced fractions with different activities, all of which were much higher than the source hydrolysate. Separation of protein hydrolysates by molecular weight and hydrophobicity clearly enhanced peptide ACE inhibitory activity, particularly purification by ultrafiltration and chromatography. The highest biological potential (and ACE inhibitory activity) among the purified peptides was observed in the ultrafiltered <1 kDa fraction, which supports the importance of high hydrophobic amino acid and proline content in ACE inhibitory peptides. These results highlight the promise of controlled protein hydrolysis with a fungal protease complex to isolate bioactive peptides from cowpea V. unguiculata proteins, which can then be further purified and/or used as an ingredient in functional foods designed for specific diets. Acknowledgements This research was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) through doctoral scholarship 164186. This research forms part of Project 25796 “Purificación y caracterización de péptidos con bioactividad antihipertensiva, antioxidante y antimicrobiana aislados de frijoles lima (Phaseolus lunatus) y caupí (Vigna unguiculata L. walp)”, financed by the CONACYT. References [1] Bougatef A, Balti R, Nedjar-Arroume N, Rovallec R, Adje EY, Souissi N, et al. Evaluation of angiotensin I-converting enzyme (ACE) inhibitory activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates generated by gastrointestinal proteases: identification of the most potent active peptide. Eur Food Res Technol 2010;231:127–35. [2] Zao XH, Li YY. An approach to improve ACE-inhibitory activity of casein hydrolysates with plastein reaction catalized by Alcalase. Eur Food Res Technol 2009;229:795–805. [3] Akillioglu HG, Karakaya S. Effects of heat treatment and in vitro digestion on the angiotensin converting enzyme inhibitory activity of some legume species. Eur Food Res Technol 2009;229:915–21.

872

M.R. Segura-Campos et al. / Process Biochemistry 46 (2011) 864–872

[4] Mao XY, Ni JR, Sun WL, Hao PP, Fan L. Value-added utilization of yak milk casein for the production of angiotensin I-converting enzyme inhibitory peptides. Food Chem 2007;103:1282–7. [5] Ma MS, Bae IY, Lee HG, Yang CB. Purification and identification of angiotensin Iconverting enzyme inhibitory peptide from buckwheat (Fagopyrum esculentum Moench). Food Chem 2006;96:36–42. [6] Zhenbao Z, Nongxue Q, Jianhua Y. Production and characterization of angiotensin converting enzyme (ACE) inhibitory peptides from apricot (Prunus armeniaca L.) kernel protein hydrolysate. Eur Food Res Technol 2010;231(1):13–9. [7] Wijesekara I, Kim SK. Angiotensin-I-converting enzyme (ACE) inhibitors from marine resources: prospects in the pharmaceutical industry. Mar Drugs 2010;8:1080–93. [8] Jenn-Shou T, Tai-Jung C, Bonnie Sun P, Shein-Da G, Mei-Yuh C. Antihypertensive effect of bioactive peptides produced by preotease-facilitated lactic acid fermentation of milk. Food Chem 2008;106(2):552–8. [9] Freitas RL, Teixeira AR, Ferreira RB. Characterization of the proteins from Vigna unguiculata seeds. J Agric Food Chem 2004;52(6):1682–7. [10] Segura Campos MR, Chel Guerrero LA, Betancur Ancona DA. Angiotensin-I converting enzyme inhibitory and antioxidant activities of peptide fractions extracted by ultrafiltration of cowpea Vigna unguiculata hydrolysates. J Sci Food Agric 2010;90:2512–8. [11] Kim JM, Whang JH, Suh HJ. Enhancement of angiotensin I converting enzyme inhibitory activity and improvement of the emulsifying and foaming properties of corn gluten hydrolysate using ultrafiltration membranes. Eur Food Res Technol 2004;218:133–8. [12] Wang W, Gonzalez de Mejia E. A new frontier in soy bioactive peptides that may prevent age-related chronic diseases. Comp Rev Food Sci Food Safety 2005;4:63–78. [13] Betancur-Ancona D, Gallegos-Tintoré S, Chel-Guerrero L. Wet fractionation of Phaseolus lunatus seeds: partial characterization of starch and protein. J Sci Food Agric 2004;84:1193–201. [14] Nielsen P, Petersen D, Dammann C. Improved method for determining food protein degree of hydrolysis. J Food Sci 2001;66(5):642–6. [15] Cho MJ, Unklesbay N, Hsieh F, Clarke AD. Hydrophobicity of bitter peptides from soy protein hydrolysate. J Agric Food Chem 2004;52(19):5895–901. [16] Hayakari M, Kondo Y, Izumi H. A rapid and simple espectrophotometric assay of angiotensin-converting enzyme. Anal Biochem 1978;84:361–9. [17] Megías C, Yust MM, Pedroche J, Lquari H, Girón-Calle J, Alaiz M, et al. Purification of an ACE inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L. protein isolates). J Agric Food Chem 2004;52:1928–32. [18] Alaiz M, Navarro JL, Giron J, Vioque E. Amino acid analysis by high-performance liquid chromatography after derivatization with diethylethoxymethylenemalonate. J Cromatogr 1992;591:181–6. ˜ y análisis de experimentos. México, D.F.: Limusa-Wiley; [19] Montgomery D. Diseno 2004. [20] Wanasundara P, Amarowikz R, Pegg RB, Shand PJ. Preparation and characterization of hydrolyzed proteins from defibrinated bovine plasma. Food Chem Toxicol 2002;67:623–30. [21] Clemente A, Vioque J, Sánchez-Vioque R, Pedroche J, Millán F. Production of extensive Chickpea (Cicer arietinum L.) protein hydrolysates with reduced antigenic activity. J Agric Food Chem 1999;47:3776–81. [22] Segura-Campos MR, Espinosa-García L, Chel-Guerrero LA, Betancur-Ancona D. Effect of enzymatic hydrolysis on solubility, hydrophobicity and in vivo digestibility in cowpea (Vigna unguiculata). Int J Food Prop, in press. [23] Imm JY, Lee CM. Production of seafood flavor from red hake (Urophycis chuss) by enzymatic hydrolysis. J Agric Food Chem 1999;47:2360–6. [24] Rebeca B, Pena-Vera MT, Diaz-Castaneda. Production of fish protein hydrolysates with bacterial proteases; yield and nutritional value. J Food Sci 1991;56:309–14. [25] Hernández Ledesma B, Amigo L, Ramos M, Recio I. Angiotensin-converting enzyme inhibitory activity in commercial fermented products. Formation

[26]

[27] [28]

[29] [30]

[31]

[32]

[33]

[34]

[35] [36]

[37]

[38]

[39]

[40]

[41]

[42]

[43] [44] [45] [46]

of peptides under simulated gastrointestinal digestion. J Agric Food Chem 2004;52:1504–10. Je JY, Park PJ, Kwon JY, Kim SK. A novel angiotensin I converting enzyme inhibitory peptide from Alaska Pollack (Theragra chalcogramma) frame protein hydrolysate. J Agric Food Chem 2004;52:7842–5. Korhonen H, Pihlanto A. Food-derived bioactive peptides—opportunities for designing future foods. Curr Pharm Des 2003;9:1297–308. Fujita H, Yamagami T, Ohshima K. Effects of an ACE inhibitory agent, katsuobushi oligopeptide, in the spontaneously hypertensive rat and in borderline and mildly hypertensive subjects. Nutr Res 2001;21:1149–58. Hwang JS, Ko WC. Angiotensin I-converting enzyme inhibitory activity of protein hydrolysates from tuna broth. J Food Drug Anal 2004;12(3):232–7. Cao W, Zhang C, Hong P, Ji H, Hao J. Purification and identification of an ACE inhibitory peptide from the peptic hydrolysate of Acetes chinensis and its antihipertensive effects in spontaneously hypertensive rats. Int J Food Sci Technol 2010;45:959–65. Wang L, Xu H, He X, Wang X. Studies on the preparation of ACE inhibitory peptides from konjac fly powder by alkaline protease enzymolysis. J Chinese Inst Food Sci Technol; 2010. DOI. CNKI: SUN: ZGSP.0.2010-01-008. Ji-Eun K, Ki H, Sam-Pin L. ACE inhibitory and hydrolytic enzyme activities in textured vegetable protein in relation to the solid state fermentation period using Bacillus subtilis HA. Food Sci Biotechnol 2010;19(2):487–95. Quirós A, Hernández-Ledesma B, Ramos M, Amigo L, Recio I. Angiotensinconverting enzyme inhibitory activity of peptides derived from caprine kéfir. J Dairy Sci 2005;88:3480–7. Lee JK, Lee MS, Park HG, Kim SK, Byun HG. Angiotensin I converting enzyme inhibitory peptide extracted from freshwater zooplankton. J Med Food 2010;13(2):357–63. Pozo-Bayón MA, Alcaíde JM, Polo MC, Pueyo E. Angiotensin I converting enzyme inhibitory compounds in white and red wines. Food Chem 2007;100:43–7. Wu J, Aluko RE, Nakai S. Structural requirements of angiotensin I-converting enzyme inhibitory peptides: quantitative structure-activity relationship study of di- and tripeptides. J Agric Food Chem 2006;54:732–8. Cheung HS, Wang FL, Ondetti MA, Sabo EF, Cushman DW. Binding of peptide substrates and inhibitors of angiotensin I-converting enzyme. J Biol Chem 1980;255:401–7. Kawasaki T, Seki E, Osajima K, Yoshida M, Asada K, Matsui T, et al. Antihypertensive effect of valyltyrosine, a short chain peptide derived from sardine muscle hydrolyzate, on mild hypertensive subjects. J Hum Hypertens 2000;14:519–23. Matsufuji H, Matsui T, Seki E, Osajima K, Nakashima M, Osajima Y. Angiotensin I-converting enzyme inhibitory peptides in an alkaline protease hydrolysate derived from sardine muscle. Biosci Biotechnol Biochem 1994;58:2244–5. Seki E, Kawasaki T, Yoshida M, Osajima K, Tamaya K, Matsui T, et al. Antihypertensive effect of sardine peptide and Val-Tyr in spontaneously hypertensive rats. J Jpn Soc Nutr Food Sci 1999;52:271–7. Matsui T, Tamaya K, Seki E, Osajima K, Matsumoto K, Kawasaki T. Absorption of Val-Tyr with in vitro angiotensin I-converting enzyme inhibitory activity into the circulating blood system of mild hypertensive subjects. Biol Pharm Bull 2002;25:1228–30. Matsui T, Hayashi A, Tamaya K, Matsumoto K, Kawasaki T, Murakami K, et al. Depressor effect induced by dipeptide, Val-Tyr, in part, to the suppression of human circulating renin–angiotensin system. Clin Exp Pharm Phys 2003;30:262–5. Moskowitz DW. Is ‘somatic’ angiotensin I-converting enzyme a mechanosensor? Diabetes Technol Ther 2003;4:841–58. Clare DA, Swaisgood HE. Bioactive milk peptides: a prospectus. J Dairy Sci 2000;83:1187–95. Meisel H. Biochemical properties of regulatory peptides derived from milk proteins. Biopolymers 1997;43:119–28. Hong LG, Wei L, Liu H, Hui SY. Mung-bean protein hydrolysates obtained with alcalase exhibit angiotensin I-converting enzyme inhibitory activity. Food Sci Technol Int 2005;11(4):281–7.