Process Biochemistry 49 (2014) 16911698

Contents lists available at ScienceDirect

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

ACE inhibitory, hypotensive and antioxidant peptide fractions from

Mucuna pruriens proteins
Francisco Gilberto Herrera Chal a , Jorge Carlos Ruiz Ruiz b ,
Juan Jos Acevedo Fernndez c,1 , David Abram Betancur Ancona a ,
Maira Rubi Segura Campos a,
a
Facultad de Ingeniera Qumica, Universidad Autnoma de Yucatn, Perifrico Norte, Km. 33.5, Tablaje catastral 13615, Col. Chuburn de Hidalgo Inn,
Mrida, Yucatn CP 97203, Mexico
b
Departamento de Ingeniera Qumica-Bioqumica, Instituto Tecnolgico de Mrida, Av. Tecnolgico, Km. 4.5 S/N, Mrida, Yucatn 97118, Mexico
c
Facultad de Medicina de la Universidad Autnoma del Estado de Morelos, Calle Iztaccihutl Esq, Leneros S/N, Volcanes, Cuernavaca, Morelos 62350, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Hydrolysates and peptide fractions obtained from Mucuna pruriens protein concentrate were stud-
Received 21 February 2014 ied for their angiotensin converting enzyme (ACE) inhibitory, hypotensive and antioxidant activities.
Received in revised form 3 June 2014 The hydrolysate obtained by pepsinpancreatin (HPP) was the most active with an ACE IC50 value of
Accepted 26 June 2014
19.5 g/mL, a Trolox equivalent antioxidant capacity (TEAC) value of 102.8 mM/mg and a ferric reducing
Available online 5 July 2014
power (FRP) IC50 of 67.2 g/mL. At a dose of 5 mg/kg HPP decrease systolic (32.2%) and diastolic (37%)
blood pressure in rats more pronounced than Captopril. The peptide fraction <1 kDa from HPP was the
Keywords:
most active with an ACE inhibitory of 10.2 g/mL (IC50 ), a TEAC value of 709.8 mM/mg and a FRP IC50
Mucuna pruriens
Hydrolysates
of 54.9 g/mL. These results indicate that the hydrolysates and peptide fractions of M. pruriens would
Peptide fractions be used as nutraceuticals ingredients for preventing and providing therapy against hypertension and
Hypotensive diseases related to oxidative damage.
Antioxidant 2014 Published by Elsevier Ltd.

1. Introduction
The renin angiotensin system plays an important role in blood
pressure, and in cardiac and vascular functions. Renin produces
decapeptide Angiotensin I from angiotensinogen. The angiotensin
I-converting enzyme (ACE) catalyzes the formation of angiotensin
II by cleaving the dipeptide from the C-terminal of angiotensin I
in the vascular wall. Therefore, the inhibition of ACE activity is a
good target for antihypertension [1]. ACE inhibitors have been pre-
scribed for hypertensive patients throughout the world, and many
clinical application data have demonstrated that ACE inhibitors
signicantly reduce the morbidity and mortality of patients with
myocardial infarction or heart failure. However, ACE inhibitors
may induce skin rashes, angioneurotic edema, diarrhea, cough, and
dizziness [2]. Because hypertensive patients often need life-long
medical treatment, interest has been focused on the isolation and
identication of ACE-inhibitory which may be obtained from new
and varied sources like foods.
Corresponding author. Tel.: +52 999 946 09 56; fax: +52 999 946 09 94.
E-mail address: maira.segura@uady.mx (M.R. Segura Campos).
1
Tel.: +52 01 777 329 70 48.
Aerobic organisms must deal with free radicals that are gen-
erated from sequential reduction of oxygen during the normal
course of aerobic metabolism. These radicals may cause cellular
damage leading to a number of pathological conditions, including
atherosclerosis, arthritis, insulin resistance, diabetes, and carcino-
genesis, if produced in an uncontrolled manner [3]. In addition,
radical mediated oxidation of fats and oils is also of a great concern
in the food industry, as it leads to the development of undesirable
off-avors and potentially toxic reaction products [4]. Currently,
synthetic antioxidants such as butylated hydroxyanisole (BHA) and
butylated hydroxytoluene (BHT) are commonly used in food. How-
ever, the potential adverse effects of these synthetic additives have
stimulated their replacement by natural antioxidants derived from
dietary sources [5].
In recent years, peptides from partial enzymatic hydrolysates
of food proteins have received a greater attention from food scien-
tists than ever before. Many biological peptides promoting health
benets have been classied and identied from food protein
hydrolysates. These peptides are inactive within the sequence of
the parent protein, but can be released during enzymatic diges-
tion or food processing. Great accomplishments have already been
achieved and some of these peptides have already been marketed
in Japan [5]. Mucuna pruriens bean is an underutilized tropical

http://dx.doi.org/10.1016/j.procbio.2014.06.021
1359-5113/ 2014 Published by Elsevier Ltd.
1692 F.G. Herrera Chal et al. / Process Biochemistry 49 (2014) 16911698
legume grown in Africa, South America and South Asia as a green
manure/cover crop. It is rich in protein (2335%) and has a nutri-
tional quality comparable to that of other pulses like soybean and
lima bean. It has good potential as a cheap and alternate source of
protein [6].
Alkaline extraction is a technological alternative for protein iso-
lation from beans. The isolated protein has low trypsin inhibitor
activity and generally meets FAO recommendations for essential
amino acids content in diets for adults [7]. Processing M. pruriens
bean also reduced l-DOPA content of protein concentrate to lev-
els of 0.1% dry matter [8]. Meaning M. pruriens bean is potentially
useful as a supplementary vegetable protein source in food manu-
facturing.
The objective of this study was to evaluate the angiotensin
I-converting enzyme inhibitory, antihypertensive and antioxi-
dant activities of peptide fractions produced from enzymatic
hydrolysates of M. pruriens bean protein concentrate.
2. Materials and methods
2.1. Grains and chemicals
Pods of M. pruriens were collected in Yucatan, Mexico. After thor-
oughly drying, the pods were thrashed to remove grains. The grains,
after thorough clearing and removal of broken grains, foreign mate-
rials and immature grains, matured and dried grains were stored
in airtight plastic jars at room temperature (25 C). All chemicals
were reagent grade or better and purchased from Sigma Chemical
Co. (St. Louis, MO, USA).
2.2. Protein concentrate
Selected grains were ground in a disk mill (model 4-E Quaker,
Mill Straub Co., Philadelphia, PA, USA) and then sifted through 4.76
and 2.38 mm screens in order to remove the smallest particles
before the air classication. Hulls were removed with a uidizing
air bed and the our resulted was milled in a Cyclotec mill (Tecator,
Hganas, Sweden) until passing through a 0.841 mm screen. The
protein concentrate of M. pruriens bean was obtained by wet frac-
tionation [9]. Briey, 5.0 kg M. pruriens bean our was suspended
in 3% sodium bisulphite in a 1:6 (w:v) ratio, pH was adjusted to
8 using 1.0 M NaOH and the suspension left to soak under con-
stant agitation for 1 h. The suspension was then passed through a
0.177 mm screen to separate the ber solids from the protein and
starch containing liquid portion. Residual solids were washed three
times with 300 mL of 3% sodium bisulphite. The suspension left to
sediment for 30 min to recover the starch after separation of the
solubilized protein. The pH of the protein solution was adjusted to
4.2 with 1.0 M HCl. The suspension was centrifuged at 1317 g for
20 min (Mistral 3000i, Curtin Matheson Sci., Houston, TX, USA), the
precipitate was freeze-dried at 47 C and 13 103 mbar (Free-
Zone 4.5, Labconco, Kansas City, MO, USA), pulverized and stored
until required.
2.3. Enzymatic hydrolysis
Hydrolysis of the protein isolates was done using a totally
randomized design. Treatments were the sequential enzymatic sys-
tem applied: Alcalase 2.4L FG and Flavourzyme 500MG (Novo
Nordisk, Bagsvaerd, Denmark); or pepsin from porcine gastric
mucosa (Sigma, P7000-100G) and pancreatin from porcine pan-
creas (Sigma, P3292-100G). The response variable was degree of
hydrolysis (DH). Hydrolysis was done under controlled condi-
tions (temperature, pH and stirring) in a 1000 mL reaction vessel
equipped with a stirrer, thermometer and pH electrode. The hydrol-
ysis with the Alcalase Flavourzyme system was done according
to the method proposed by Pedroche et al. [10]. Protein concen-
trate was suspended in distilled water to produce a 10% (w/v)
protein solution which was equilibrated at optimum temperature
and pH for each protease before adding the respective enzyme.
Protease was then added to the solution at a ratio of 0.3 AU/g for
Alcalase and 50 LAPU/g for Flavourzyme . Hydrolysis conditions
were 45 min at 50 C for each enzyme, using pH 8.0 for Alcalase
and pH 7.0 for Flavourzyme . The pH was kept constant by adding
1.0 M NaOH during hydrolysis. The hydrolysis with the sequen-
tial pepsinpancreatin system was done for 90 min: predigestion
with pepsin for 45 min followed by incubation with pancreatin for
45 min. Hydrolysis parameters were substrate concentration 4%;
enzyme/substrate ratio 1:10; pH 2.0 for pepsin; pH 7.5 for pancre-
atin; and 37 C hydrolysis temperature. The reaction was stopped
by heating to 80 C for 20 min, followed by centrifuging at 1317 g
for 20 min (Mistral 3000i, Curtin Matheson Sci.) to remove the insol-
uble portion.
2.4. Degree of hydrolysis
Degree of hydrolysis (DH) was calculated by determining free
amino groups with o-phthaldialdehyde [11]. DH = h/htot 100;
where htot is the total number of peptide bonds per protein equiv-
alent, and h is the number of hydrolyzed bonds. The htot factor is
dependent on raw material amino acid composition. For hard to
cook protein isolated htot = 7.66 mmol/g of protein. Determinations
were made by triplicate.
2.5. Proximate composition of protein concentrate and
hydrolysates
Proximate composition of the M. pruriens protein concentrate
and the derived protein hydrolysates was calculated using of-
cial AOAC procedures [12]. Nitrogen (method 954.01); fat (920.39);
ash (923.03); ber (962.09) and moisture (925.09). Protein content
was calculated as nitrogen 6.25, and carbohydrate content was
estimated as nitrogen-free extract (NFE). All determinations were
made by triplicate.
2.6. Hydrolysate fractionation
The Alcalase Flavourzyme (HAF) and pepsinpancreatin
(HPP) hydrolysates were fractionated by ultraltration [13] with
a high performance ultraltration cell (Model 2000, Millipore, Bil-
lerica, MA, USA). The supernatants of Alcalase Flavourzyme and
pepsinpancreatin hydrolysates were collected. Part of them was
studied as whole hydrolysates, and the rest was fractionated [13].
Five fractions were prepared using four molecular weight cut-off
(MWCO) membranes: 1 kDa, 3 kDa, 5 kDa and 10 kDa. Soluble frac-
tions were prepared by ultraltering the hydrolysates through the
MWCO membranes beginning with the largest 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 ow rate. Per-
meate from the 10 K membrane was then ltered 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 mem-
brane. The ve ultraltered peptide fractions (UFPF) from each
hydrolysis treatment (HAF or HPP) were prepared and designated
as >10 kDa (10 kDa retentate); 510 kDa (10 kDa permeate5 kDa
retentate); 35 kDa (5 kDa permeate3 kDa retentate); 13 kDa
(3 kDa permeate1 kDa retentate); and <1 kDa (1 kDa permeate).
Whole hydrolysates and peptide fractions were freeze-dried and
stored at 20 C until analysis.
 F.G. Herrera Chal et al. / Process Biochemistry 49 (2014) 16911698
2.7. Protein determination in fractions
Protein content was measured using the Folin reagent method
[14]. In this assay, the peptide nitrogen in the peptide fractions
reacts with the copper [II] ions under alkaline conditions and
the subsequent reduction of the FolinCiocalteu phosphomolyb-
dic phosphotungstic acid to heteropolymolybdenum blue by the
copper-catalyzed oxidation of aromatic acids. Bovine serum albu-
min (1 mg/mL) was used as standard to generate a curve from 0 to
1 mg/mL. Determinations were made by triplicate.
2.8. Amino acid composition
Amino acid composition was determined for the protein
concentrates, hydrolysates peptide fractions (<1 kDa) by high per-
formance liquid chromatography [15]. Samples (4 mg of protein)
were treated with 4 mL of HCl 6.0 N, placed in hydrolysis tubes
and gassed with nitrogen at 110 C for 24 h. They were then dried
in a rotavapor (Bchi, Rotavapor R-215, Flawil, Switzerland) and
suspended in sodium borate buffer (1.0 M, pH 9.0). Derivatization
was performed at 50 C using diethyl ethoxymethylenemalonate.
Amino acids were separated using HPLC with a reversed-phase
column (300 mm 3.9 mm, Nova Pack C18, 4 mm; Waters), and
a binary gradient system with sodium acetate containing 25 mM
(A) 0.02 g/L sodium azide at pH 6.0, and (B) acetonitrile as sol-
vent. The ow-rate was 0.9 mL/min, and the elution gradient was:
time 0.03.0 min, linear gradient A:B (91:9) to AB (86:14); time
3.013.0 min, elution with AB (8614); time 13.030.0 min, linear
gradient AB (86:14) to AB (69:31); time 30.035.0 min, elution
with AB (69:31). Determinations were made by triplicate.
2.9. Biological activities
The protein hydrolysates and their corresponding ultraltered
peptide fractions were analyzed to identify their ACE inhibitory,
antihypertensive and antioxidant activities.
2.9.1. ACE inhibitory activity
Angiotensin I-converting enzyme inhibitory activity in the
hydrolysates and their UFPF was analyzed following Hayakari and
Kondo [16]. Hippuryl-l-histidyl-l-leucine (HHL) is hydrolyzed by
ACE 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
the reaction terminated by adding 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 done in triplicate. ACE inhibitory activity
was quantied by a regression analysis of ACE inhibitory activity
(%) versus peptide concentration and dened as an IC50 value, that
is, the peptide concentration (g protein/mL) required to produce
50% ACE inhibition under the described conditions. Determinations
were made by triplicate.
2.9.2. Hypotensive effect in normotensive rats
The experiment described has been carried out in accordance
with the EU Directive 2010/63/EU for animal experiments. Wistar
female rats of 8- to 10-week-old and weighing 280 40 g were
used to evaluate the hypotensive activity of the following prod-
ucts: HPP (5, 10 and 15 mg/kg) and HAF (5, 10 and 15 mg/kg). They
remained at a temperature of 23 C with 12 h light/dark cycles, and
consumed tap water and a standard diet for rats (nu3lab, Research
1693
Global Solution. Brulington, Ontario, Canada) ad libitum during the
experiments. All the above-mentioned products were administered
to the rats by intraperitoneal injection (IP), between 9 and 10 a.m.
Four animals were used per treatment. Physiological saline solu-
tion (PSS) (Sigma, USA H4034) served to dilute hydrolysates and
as positive control, and Captopril (5, 10 and 15 mg/kg) (Sigma,
USA C4042), a known ACE inhibitor, served as negative control.
Hypotensive effect of the hydrolysates was determined noninva-
sively following the method reported by Miguel et al. [17]. The
systolic blood pressure (SBP) and diastolic blood pressure (DBP) of
the rats were monitored by the tail cuff method before administra-
tion and also 2.5 h post-administration. Before the measurement,
the rats were anesthetized with a single dose (30 mg/kg, IP) of
sodium barbital (Sigma, USA B0375) and kept at 30 C for 30 min
to make the pulsations of the tail artery detectable. The original
method for measuring arterial blood pressure using the tail cuff
provides SBP and DBP values detected by a physiographer (CPM
Physiograph Narco Bio-System Inc., Houston, TX; CODA and RTBP
2000, Kent Scientic, and 7D Polygraph, Grass Instruments). The
blood pressure signals were acquired (at 1.0 kHz) on a computer
using the analog-digital converter Mini Digi B (Axon Instruments)
and software AxoScope 10.2. To analyze the blood pressure reading,
was used a 10.2 Clampt (Axon Instruments). Blood pressure was
recorded for at least 2.5 h for each rat. In this period, the rst 30 min
represent basal blood pressure and then administered via IP evalu-
ate treatments: negative control (PSS), positive control (Captopril )
and hydrolysates. To minimize stress-induced variations in blood
pressure, all measurements were taken by the same person in the
same peaceful environment. Moreover, to guarantee the reliabil-
ity of the measurements, we established a training period of two
weeks before the actual trial time, and during this period the rats
were accustomed to the procedure. Determinations were made by
triplicate. The percentage of hypotensive effect was calculated from
the following formula:
PA Exp
Hypotensive effect (%) = 100
PA Ctrl
where PA Exp is the SBP and DBP observed after administration
of PSS, Captopril , and hydrolysates and PA Ctrl is the basal blood
pressure observed during the rst 30 min registration.
2.9.3. Trolox equivalent antioxidant capacity
The ABTS+ radical cation was produced by reacting 2,2 -
azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) with
potassium persulfate [18]. To prepare the stock solution, ABTS was
dissolved at a 2 mM concentration in 50 mL phosphate-buffered
saline (PBS) prepared from 4.0908 g NaCl, 0.1347 g KH2 PO4 ,
0.7098 g Na2 HPO4 , and 0.0749 g KCl dissolved in 500 mL ultra-
pure water. If pH was lower than 7.4, it was adjusted with NaOH.
A 70 mM K2 S4 O8 solution in ultrapure water was prepared. The
ABTS radical cation was produced by reacting 10 mL ABTS stock
solution with 40 L K2 S4 O8 solution and allowing the mixture
to stand in darkness at room temperature for 1617 h before
use. The radical was stable in this form for more than 2 days
when stored in darkness at room temperature. Antioxidant com-
pound content in hydrolysates and peptide fractions were analyzed
by diluting the ABTS+ solution with PBS to an absorbance of
0.800 0.030 AU at 734 nm. After adding 990 L diluted ABTS+
solution (A 734 nm = 0.800 0.030) to 10 L of 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (TROLOX) standard (nal
concentration 0.53.5 mM) in PBS, absorbance was read at room
temperature exactly 6 min after initial mixing. All analyses were
run in triplicate. The percentage decrease in absorbance at 734 nm
was calculated and plotted as a function of the antioxidant con-
centration of Trolox for the standard reference data. To calculate
the Trolox equivalent antioxidant capacity (TEAC), the slope of the
1694 F.G. Herrera Chal et al. / Process Biochemistry 49 (2014) 16911698
absorbance inhibition percentage versus antioxidant concentration
plot was divided by the slope of the Trolox plot. This produces
the TEAC at a specic point in time. Determinations were made
by triplicate.
2.9.4. Ferric reducing antioxidant power assay
This method is based on the reduction of potassium ferricyanide
(Fe3+ ) to Fe2+ in the presence of an antioxidant, KFeIII forming the
blue complex [FeII (CN6 )], which absorbing a 700 nm [19]. First,
200 L of sample (containing 1 mg of protein), 500 L of phosphate
buffer (0.2 M, pH 6.6), and 500 L of potassium ferricyanide (1%)
were mixed in a test tube. The test tube was then incubated at
50 C for 20 min. Subsequently, 500 mL of trichloroacetic acid (10%)
were added, and the tube was centrifuged at 3000 g for 10 min. An
aliquot of 500 L of the supernatant, which is dissolved in an equal
amount of distilled water, immediately 500 L of ferric chloride
(0.1%) were added. Absorbance was determined at 700 nm. Samples
were tested in a range of concentrations from 200 to 1000 mg/mL.
Butylated hydroxytoluene was used as control in the same range
of concentrations. Determinations were made by triplicate.
2.10. Statistical analysis
All results were analyzed using central tendency and dispersion
measures. One-way ANOVAs were run to evaluate protein isolate
hydrolysis data and ACE inhibitory and antioxidant activities. A LSD
multiple range test was used to determine differences between
treatments. All analyses were done according to Montgomery [20]
and processed with the Statgraphics Plus version 5.1 software.
3. Results and discussion
3.1. Degree of hydrolysis (DH)
Both sequential enzymatic systems produced high DH
(Alcalase Flavourzyme = 20.23%; pepsinpancreatin = 40.15%),
although they differed (p < 0.05). These values were similar than
those reported for hydrolysis of other legumes like Vigna ungui-
culata (53.1, 58.8 and 35.7%) with Alcalase , Flavourzyme and
pepsinpancreatin for 90 min [21], and of Phaseolus vulgaris (43.1
and 26.2%) with Alcalase Flavourzyme and pepsinpancreatin
for 90 min [22]. Variation in DH values was probably the result of
enzymatic system hydrolytic specicity. Peptides with biological
activities have generally been isolated from food proteins via
hydrolysis with digestive enzymes such as pepsin, pancreatin,
or chymotrypsin [10]. In comparison to animal or plant derived
enzymes, microbial proteases such as Alcalase , Protamex, and
Flavourzyme have also shown excellent potential to produce
highly biological and functional hydrolysates [23].
3.2. Proximate composition
Table 1 presents the proximate composition (dry weight) of
the M. pruriens protein concentrate and hydrolysates. The mois-
ture content was in the range of 7.0617.58%. The moisture content
was related to the kind of sample and to the method employed for
removing the water. The protein concentrate of M. pruriens and its
hydrolysates were freeze-drying; however both hydrolysates had
moisture content twice than the protein concentrate. The higher
moisture content observed in the hydrolysates was due to its hygro-
scopic characteristics.
The crude protein contents of the M. pruriens protein con-
centrate and its hydrolysates were 51.27, 38.92 and 45.89%,
respectively. Protein content is lower in the hydrolysates due to
the hydrolysis process included a step to remove the insoluble
Table 1
Proximate composition (% d.b.) M. pruriens protein concentrate and hydrolysates.
Component Protein Hydrolysates
concentrate
Alcalase Flavourzyme Pepsinpancreatin
Moisture (7.06 0.39 )
a
(17.58 0.33 )
c
(13.08 0.28b )
Protein 51.27 0.13c 38.92 0.41a 45.89 0.57b
Fat 14.45 0.07c 13.30 0.50b 9.93 0.08a
Crude ber 1.72 0.17b 1.48 0.08a 1.37 0.03a
Ash 13.78 0.04a 19.18 0.05b 22.69 0.02c
NFE 18.78 0.34a 37.12 0.79c 20.13 0.48b
ac
Different superscript letters in the same row indicate statistical difference
(p < 0.05). NFE, nitrogen free extracts.
portion of the hydrolysates. The results obtained in this study indi-
cate that soluble protein content in the hydrolysates was related
to the degree of hydrolysis. Hydrolysis generated polypeptides,
peptides and amino acids that are more soluble than the native
protein. The hydrolysate obtained with pepsinpancreatin showed
a higher degree of hydrolysis compared to the hydrolysate obtained
with Alcalase Flavourzyme , so that its protein content was
higher. Hydrolysates contained 13.30% and 9.93% fat content and
the difference was signicance between protein concentrate and
hydrolysates. The fat content was found to decrease with increase
in degree of hydrolysis. According to Benjakul and Morrissey [24]
lipids are partially removed together with the insoluble undigested
non-protein substances after hydrolysis. Crude ber content was
low in the protein concentrate and its hydrolysates. Processes used
for the obtaining of the protein concentrate and its hydrolysates
include stages for removal of brous residues. These processes
could have caused low contents of crude ber. The higher ash con-
tent of hydrolysates was due to the addition of alkali required for pH
adjustment and its control during the hydrolytic process. Accord-
ing to Severin and Xia [25], as degree of hydrolysis increase, the pH
of the hydrolysis process decrease and subsequently the volume
of NaOH used will also increase. The higher content of Nitrogen-
Free Extract (NFE) in the hydrolysates, compared with the protein
concentrate, was due to the low content of protein and fat in both
samples.
3.3. Hydrolysate fractionation
Hydrolysates from both sequential enzymatic systems
were fractionated using four membranes with different
MWCO. The resulting fractions were designated as >10, 510,
35, 13 and <1 kDa. The peptide fractions obtained with
Alcalase Flavourzyme system had a protein content ran-
ging between 0.348 and 0.986 mg/mL, the protein content did
not decrease in proportion as the MWCO decreased, the frac-
tion 35 kDa exhibit the highest content of protein. The protein
content of peptide fractions from the hydrolysate obtained with
pepsinpancreatin ranged between 0.088 and 1.813 mg/mL. In this
system the protein content decreased proportionally according to
the MWCO membranes employed, showing the greatest amount
of protein in the fraction >10 kDa and the lower in fraction <1 kDa.
3.4. Amino acid composition
Table 2 summarizes the amino acid content in protein concen-
trate, HAF, HPP and peptide fractions (<1 kDa). Amino acid analysis
of M. pruriens protein concentrate contained a good proportion
of hydrophobic amino acids. Protease specicity is an important
factor to be considered to prepare specic peptides with various
nutraceutical functions from proteins, besides the protein source.
Alcalase and Flavourzyme are proteases for industrial use
which hydrolyses peptide bonds with broad specicity liberating
 F.G. Herrera Chal et al. / Process Biochemistry 49 (2014) 16911698 1695

Table 2
Comparison of amino acid contents of M. pruriens protein concentrate (PC) Alcalase Flavourzyme hydrolysate (HAF), pepsinpancreatin hydrolysate (HPP) and peptide
fractions (<1 kDa).

Amino acid PC HAF HPP HAF (F < 1 kDa) HPP (F < 1 kDa)

+Asp 15.79b 19.33d 17.71c 20.03e 14.35a

++Glu 10.52a 10.89a 12.87b 12.51b 13.77c
Ser 12.49b 8.66a 14.33c 14.80c 7.89a
His 8.07c 10.87d 5.22b 5.32b 3.47a
Gly 5.56b 7.13c 4.98a 5.02a 4.52a
Thr 12.44d 8.20c 8.60c 7.89b 5.79a
Arg 7.24c 4.27a 5.28b 4.75a 7.67c
Ala 3.71b 2.89a 4.09c 3.67b 4.89c
Pro 0.72c 1.01e 0.66b 0.81d 0.22a
Tyr 3.33b 5.55d 3.84b 4.24c 2.40a
Val 2.71b 5.26d 2.94b 3.07c 1.57a
Met ND ND ND ND ND
Cys 3.93a 3.90a 4.30b 4.50b 6.91c
Ile 4.04b 2.93a 4.27b 4.26b 8.29c
Trp 0.77d 0.67c 0.51b 0.54b 0.26a
Leu 1.91b 2.21c 2.43c 1.32a 2.39c
Phe 0.74a 1.69b 0.80a 0.89a 8.06a
Lys 6.04b 4.53a 7.17c 6.38b 7.55c
Amino acid distribution (%)
Hydrophobic 14.6a 16.7c 15.7b 14.6a 25.7d
Hydrophilic 47.7a 49.9c 48.3b 51.9d 46.8a
Neutral 37.7c 33.4b 36.0c 33.5b 25.5a

Hydrophobic (Ala, Val, Met, Phe, Leu, Ile, Pro, Trp).

Hydrophilic (Arg, Asp, His, Lys, Glu)
Neutral (Ser, Gly, Thr, Tyr, Cys)
+Aspartic acid + asparagine.
++Glutamic acid + glutamine.
ND = no detected.
ac
Different superscript letters in the same row indicate statistical difference (p < 0.05).

peptides with hydrophobic amino acids such as Phe, Tyr, Trp,
Leu, Ile, Val and Met at their C-terminal [26]. Mean while pepsin
and pancreatin are proteases with more specic, which mainly
generates oligopeptides (6070%) and free amino acids (3040%)
[27]. In this respect, both enzymatic systems were very suitable for
the production of ACE inhibitory and antioxidant peptides and the
M. pruriens protein concentrate was a good source of such bioac-
tive peptides upon hydrolysis by commercial and gastrointestinal
enzymes as conrmed by this study.
Comparison of the amino acid compositions and the biologi-
cal properties reveal that hydrolysates and ultraltered peptide
fractions (<1 kDa) are most active than protein concentrate, appar-
ently because have an abundance of hydrophobic and hydrophilic
amino acids. According to Hwang and Ko [28] the presence in
hydrolysates of peptides with aromatic (Phe, Tyr and Trp) residues
at its C-terminal and basic (Lys, His, Arg) or hydrophobic ones at
its N-terminal were essential for strong and competitive inhibi-
tion on ACE. For antioxidant mechanism some amino acids were
widely believed to be direct radical scavengers due to their special
groups in side chains, such as His (imidazole group) [4], Trp (indolic
group), and Tyr (phenolic group). These groups could act as hydro-
gen donors. Additionally, Cys donates the sulfur hydrogen [29].
Aromatic amino acids (Tyr and Phe) are generally considered as
effective radical scavengers, because they can donate protons eas-
ily to electron decient radicals while at the same time maintaining
their stability via resonance structures [30].
3.5. Biological activities
3.5.1. ACE inhibitory activity
The hydrolysate concentration required to produce 50% inhibi-
tion of ACE (IC50 ) was used as an activity indicator. This indicator
was expressed as g protein/mL, with smaller values indicating
greater ACE inhibiting power. The non-hydrolyzed M. pruriens
protein concentrate showed no inhibitory activity on ACE. ACE
inhibitory activity was generated from the M. pruriens protein after
enzymatic hydrolysis. The IC50 value for Alcalase Flavourzyme
hydrolysate was 76.1 g/mL and that for pepsinpancreatin was
19.5 g/mL. The IC50 values registered here were lower than that
reported for enzymatic hydrolysates from different protein sources
(IC50 = 200246,000 g/mL) [3134]. Many of these hydrolysates
also have exhibited antihypertensive activity in spontaneously
hypertensive rats (SHR). The IC50 values for the HAF and HPP were
outside this range because they require fewer quantities to inhibit
50% of ACE activity. Hydrolysis released ACE-inhibitors peptides
from an inactive form within the sequence of M. pruriens pro-
tein. On the other hand, both hydrolysates had good solubility in
water, which could allow their incorporation into food matrices to
improve its functional properties and nutritional value. Ultraltra-
tion of the hydrolysates produced peptide fractions that differ in
their bioactivity (Fig. 1).
For the peptide fractions from hydrolysate obtained with
Alcalase Flavourzyme the IC50 values ranged from 54.6
to 31.3 g/mL, while the peptide fractions from hydrolysate
obtained with pepsinpancreatin had values ranging from 65.7
to 10.2 g/mL. Peptide fractions of Alcalase Flavourzyme sys-
tem exhibited higher bioactivity than the original hydrolysate
(76.1 g/mL), with the higher IC50 in <1 kDa fraction. ACE inhibitory
activity in peptide fractions of pepsinpancreatin system was
signicantly (p < 0.05) dependent on peptide fraction molecular
weight, with the lowest activity in the >10 kDa fractions and
the highest in the 13 and <1 kDa fractions. This was similar to
the IC50 values for a peptide fraction from egg white protein
hydrolysates, which exhibited the highest ACE inhibitory activity
value (40 g/mL) in the <3 kDa fraction [35]. Fujita and Yoshikawa
[36] assessed the relative antihypertensive activities of peptides
to that of captopril (antihypertensive drug); the peptides and
the drug were orally administered to SHR rats to monitor time-
course changes of blood pressures, whereby it was evidenced that
captopril showed maximal decrease of blood pressure 4 h after
oral administration with an IC50 of 0.022 g/mL. The higher IC50
values for peptide fractions (pepsinpancreatin 13 and <1 kDa)
1696 F.G. Herrera Chal et al. / Process Biochemistry 49 (2014) 16911698
100
100 5 mg/kg 10 mg/kg 15 mg/kg
Alcalase-Flavourzyme
Pepsin-Pancreatin 80
80 70.7a

DBP reduction (%)

65.7a 61.0b
60
IC50 (mg/mL)

60 54.6b 53.2b
47.2b 44.5c
40 37.0d
40 30.4b 27.7d
31.3d 25.4c
22.7b 22.9d
22.3c 18.8c
20
20 11.6d 10.2d 3.4c
1.2a 1.2a 1.2a
0
0 PSS Captopril HAF HPP
F >10 kDa F 5-10 kDa F 3-5 kDa F 1-3 kDa F <1 kDa
Fig. 3. Decrease in diastolic blood pressure (DBP) caused in normotensive
Fig. 1. ACE inhibition IC50 (g/mL) of peptide fractions obtained by ultral- rats by the administration of physiological saline solution (PSS), Captopril ,
tration from both sequential enzymatic systems Alcalase Flavourzyme and Alcalase Flavourzyme hydrolysate (HAF), and pepsinpancreatin hydrolysate
pepsinpancreatin. ad Different superscript letters in the same series indicate sta- (HPP). The data represent the mean values SEM for 46 rats. ad Different super-
tistical difference (p < 0.05). script letters in the same series indicate statistical difference (p < 0.05).

obtained in this study were 400 times less active. A synthetic

drug will always have greater activity than a natural product like
hydrolysates or peptide fractions. However hydrolysates, peptide
fractions and single peptides have several advantages: hydrolysates
not only have biological activity but can improve the nutritional
contribution [37]; peptide fractions are constituted by sets of pep-
tides with similar molecular weights, which may act in synergy
to increase its effect on the organism [36] and single peptides
can increase their antihypertensive activity after being ingested
[36]. Most of the reported peptides that exhibit ACE inhibitory
activity have low molecular weights (approx. <12 amino acids).
This means that active peptides can be selected to a degree by
ultraltration, resulting in the production of a permeate stream
with higher ACE inhibition than the original hydrolysate [38].
This is consistent with the behavior observed in the present
study.
3.5.2. Hypotensive effect in normotensive rats
The inhibitory potencies of the peptides on ACE activity did not
always correlate with their in vivo hypotensive effects. In order to
exert a hypotensive effect in vivo, the ACE inhibitory M. pruriens
hydrolysates were intraperitoneal injected to female normotensive
rats. The hypotensive effect was evaluated by measuring changes
in SBP and DBP after single administration. Figs. 2 and 3 show the
decreases of the SBP and DBP obtained, at different doses (5, 10,
and 15 mg/kg), after the IP administration of the different prod-
ucts. Before the measurement, the rats were anesthetized with a
single dose (30 mg/kg, IP) of sodium barbital and kept at 30 C for
30 min to make the pulsations of the tail artery detectable in order
100
5 mg/kg 10 mg/kg 15 mg/kg
76.5b
80
SBP reduction (%)
to obtain blood pressure data. The physiological saline solution
(negative control), used as a vehicle to dissolve the hydrolysates
and Captopril had no signicant effect (p > 0.05), with reductions
of 0.6 and 1.2% in systolic and diastolic blood pressure, respec-
tively. According to Li et al. [39], distilled water, physiological
solutions, and non-hydrolyzed proteins do not exhibit antihyper-
tensive effect. The administration of HAF and HPP produced a
signicant decrease in the SBP and DBP in the rats (p < 0.05).
The administration of HPP (5 mg/kg) produced a blood pressure
lowering effect higher to that of HAF and Captopril (p > 0.05). The
decreases in both variables, SBP and DBP, at doses of 10 and 15 m/kg
were higher than HAF but lower than Captopril. The decrease in the
hypotensive effect observed for both hydrolysates was less pro-
nounced for HPP (p < 0.05). However, the maximal decreases in
blood pressure were caused by Captopril at the three doses eval-
uated (p > 0.05). The hypotensive effect of these products was tran-
sient and reverted 24 h after the administration. At that moment,
the values of the SBP and the DBP in the rats were, therefore, similar
to the initial values. Researchers thought that the prolonged anti-
hypertensive action of ACE inhibitors might be related to persistent
inhibition of ACE activity and Ang II concentrations of tissues, such
as vascular wall and kidneys [40]. Captopril given either orally or
intravenously lowers blood pressure in both SHR and normoten-
sive rats, and both these strains are sensitive to the prejunctional
actions of angiotensin II. This provides strong evidence that the
hypotensive action of captopril is due to the interference by the
drug with the prejunctional actions of angiotensin II [41]. Cap-
topril prevents the conversion of Angiotensin I to Angiotensin II
by inhibition of ACE, a peptidyldipeptide carboxy hydrolase. This
inhibition has been demonstrated in both healthy human subjects
and in animals by showing that the elevation of blood pressure
caused by exogenously administered Angiotensin I was attenu-
ated or abolished by Captopril. In animal studies, captopril did not
alter the pressor responses to a number of other agents, including

60 Angiotensin II and norepinephrine, indicating specicity of action

[42]. Some authors [43] investigated the role of Ang II production in
40.5b
40 the plasma and kidney, their results showed the concentration of
32.2c 31.3c
Ang II in plasma had no signicance during long-term oral admin-
23.5b 24.5b
16.6c
19.6d istration of jelly sh hydrolysates, but the concentration of Ang II
20
6.7c
in kidney signicantly decreased. So, the kidney should be the tar-
0.6a 0.6a 0.6a get site of peptides. Miguel et al. [17] indicated that ACE inhibitors
0 with different molecular structures had different metabolism path-
PSS Captopril HAF HPP
ways and tissue distribution. These characteristics determined an
Fig. 2. Decrease in systolic blood pressure (SBP) caused in normotensive ACE inhibitory effect in tissue and thus exhibited different antihy-
rats by the administration of physiological saline solution (PSS), Captopril , pertensive effects. In this study commercial and gastrointestinal
Alcalase Flavourzyme hydrolysate (HAF), and pepsinpancreatin hydrolysate
(HPP). The data represent the mean values SEM for 46 rats. ad Different super-
enzymes used to hydrolyze the proteins of M. pruriens could have
script letters in the same series indicate statistical difference (p < 0.05). generated several peptides with the capacity to inhibit ACE.
 F.G. Herrera Chal et al. / Process Biochemistry 49 (2014) 16911698
Table 3
Trolox equivalent antioxidant capacity (mM/mg) and Ferric reducing power IC50
(g/mL) of peptide fractions obtained by ultraltration from both sequential enzy-
matic systems Alcalase Flavourzyme (AF) and pepsinpancreatin (PP).
Fraction Trolox equivalent antioxidant Ferric reducing power
capacity (mM/mg) IC50 (g/mL)
AF PP AF
F > 10 kDa 30.0e 10.1e 60.8c
F 510 kDa 92.1c 222.3d 64.2a
F 35 kDa 55.6d 375.7c 56.5e
F 13 kDa 102.0b 537.9b 59.7d
F < 1 kDa 162.9a 709.8a 62.6b
ae
Different superscript letters in the same series indicate statistical difference
(p < 0.05).
3.5.3. Trolox equivalent antioxidant capacity
The radical scavenging activity of each hydrolysate was
evaluated by the ABTS+ method. Results showed that both
hydrolysates exhibit antioxidant activity. On the contrary, intact M.
pruriens protein concentrate did not showed activity. Hydrolysate
obtained with pepsinpancreatin was the most active with a
trolox equivalent antioxidant capacity value of 102.8 mM/mg,
i.e., 1 mg of pepsinpancreatin hydrolysate was able to scav-
enge the same amount of radicals as a solution of 102.8 mM
of Trolox. Hydrolysate obtained with Alcalase Flavourzyme
was less active with a TEAC value of 49.0 mM/mg. The enzy-
matic hydrolysis of protein concentrates released antioxidant
peptides that were inactive when included within the intact protein
[44]. The development of antioxidant activity following enzy-
matic hydrolysis of M. pruriens protein concentrate is in line with
these reports. For the peptide fractions from hydrolysate obtained
with Alcalase Flavourzyme the TEAC values ranged from 30.0
to 162.9 mM/mg, while the peptide fractions from hydrolysate
obtained with pepsinpancreatin had values ranging from 10.1 to
709.8 mM/mg (Table 3).
Peptide fractions of Alcalase Flavourzyme system exhibited
higher bioactivity than the original hydrolysate, with the higher
TEAC value in <1 kDa fraction. Antioxidant activity in peptide
fractions of pepsinpancreatin system was signicantly (p < 0.05)
dependent on peptide fraction molecular weight, with the low-
est activity in the >10 kDa fractions and the highest in the <1 kDa
fractions. Obviously, the results reveal the presence of antioxidant
peptides in all the fractions, and a clear tendency to concentrate in
the fractions with a lower molecular weight. It is well documented
that, although antioxidant activity is normally widely observed in
every different whole and fractionated hydrolysate, the lower most
molecular weight peptide fractions usually exhibit the strongest
bioactivity. Some authors have reported that accessibility to the
oxidant-antioxidant test systems is greater for small peptides and
amino acids than for large peptides and proteins [29]. This is con-
sistent with what was observed in this study.
3.5.4. Ferric reducing antioxidant power assay
Different studies have indicated that antioxidative activ-
ity and reducing power are related [45]. In ferric reducing
antioxidant power (FRAP) assay, the yellow color of the test
solution changes to various shades of green or blue, depend-
ing on the reducing power of each sample. The presence of
antioxidants could cause the reduction of the Fe3+ /ferricyanide
complex to the ferrous form. Therefore, measuring the forma-
tion of blue complex at 700 nm can monitor the concentration
of Fe2+ [46]. Hydrolysate obtained with Alcalase Flavourzyme
was the most active with a FRAP IC50 value of 65.7 g/mL. In
example, 65.7 g of Alcalase Flavourzyme hydrolysate was
able to reduce the 50% of Fe3+ /ferricyanide complex present in
the solution assay. Hydrolysate obtained with pepsinpancreatin
1697
was less active with a FRAP IC50 value of 67.2 g/mL. The
FRAP IC50 values of M. pruriens peptide fractions are shown in
Table 3.
For the peptide fractions from hydrolysate obtained with
Alcalase Flavourzyme the FRAP IC50 values ranged from 64.2
to 56.5 g/mL, while the peptide fractions from hydrolysate
PP obtained with pepsinpancreatin had values ranging from 67.5
67.5a to 54.9 g/mL. Peptide fractions of Alcalase Flavourzyme sys-
55.2b tem exhibited higher bioactivity than the original hydrolysate
56.7b (65.7 g/mL), with the higher IC50 in 35 kDa fraction (56.5 g/mL).
56.9b
54.9c
Antioxidant activity in peptide fractions of pepsinpancreatin sys-
tem was signicantly (p < 0.05) dependent on peptide fraction
molecular weight, with the lowest activity in the >10 kDa frac-
tions and the highest in the <1 kDa fractions. FRAP generally
measures the reducing ability against ferric ion (Fe3+ ). This indi-
cates the ability of hydrolysates to donor the electron to the free
radical [47]. Therefore, it could be concluded that M. pruriens
hydrolysates, especially peptide fractions, had the reducing power,
leading to the prevention and retardation of propagation of oxi-
dation. Some proteins from certain foods have been reported to
have the ability to scavenge active oxygen species. In addition to
food proteins, some food-protein hydrolysates have been found to
exhibit antioxidant activity [48]. According to the results obtained,
the hydrolysates and its ultraltered peptide fractions have the
ability to act as radical scavengers and electron donors. The uti-
lization of protein hydrolysates or ultraltered peptide fractions
to improve the antioxidant activity in foods presents additional
advantages over other natural antioxidants, since they also confer
nutritional value, as well as, desired functional properties. Nali-
nanon et al. [49] simulate the gastrointestinal (GI) digestion using
an in vitro model with pepsinpancreatin in order to evaluate
the impact of hydrolysis on ABTS radical-scavenging activity of
antioxidant peptides. As a result, no degradation or generation
of new antioxidant peptides took place and after ABTS radical-
scavenging activity was increased by about 712%. It was suggested
that antioxidative peptides were modied by pancreatin diges-
tion to enhance their ABTS radical-scavenging activity. In addition,
the increase of hydrophilic property of GI digests after pancreatin
treatment has been reported to favor trapping of ABTS radical of
loach protein hydrolysate [49]. Additionally, You et al. [50] reported
that GI digests had increased reducing power, chelating ability
of Cu2+ and hydroxyl radical-scavenging activities by 77%, 12%
and 12%, respectively. Several studies demonstrated that a peptide
might exhibit several biological activities acting as an antioxi-
dant or as antihypertensive simultaneously. It has also reported
that the biological activity can be increased after partial hydroly-
sis.
Ultraltered peptide fractions used instead of puried pep-
tides for the development of functional foods presents multiple
advantages. Ultraltered peptide fractions are formed by groups
of peptides which could exhibit different biological activities and
may act in synergism thus increasing their biological effects on
the organism. Membrane technology that works without the addi-
tion of chemicals, with a relatively low use of energy, it has low
processing costs, the scale-up is an easy subject and the process
lines are well arranged make it the ideal technology for use on an
industrial. In addition, membrane processes are especially suitable
for the food industry, because of the mild working conditions, rela-
tively easy scale up and low processing costs in comparison to other
techniques like chromatographic.
4. Conclusions
ACE inhibitory, hypotensive and antioxidant activities have
been recorded in M. pruriens protein hydrolysates and ultraltered
peptide fractions produced with the sequential enzymatic systems
1698 F.G. Herrera Chal et al. / Process Biochemistry 49 (2014) 16911698
Alcalase Flavourzyme and pepsinpancreatin. Hydrolysates
obtained with gastrointestinal enzymes exhibit greater biological
activities; this enzyme system probably generated peptides that
can act in vitro as ACE inhibitors and effective radical scaven-
gers. It was conrmed by single short term administration that
hydrolysates at low doses decreases SBP and DBP in rats and ACE
inhibitory peptides derived from M. pruriens are closely involved
in the hypotensive effect. Although further research is needed on
purication steps and peptide sequences, hydrolysates and ultra-
ltered peptide fractions of M. pruriens are promising natural ACE
inhibitors and antioxidant peptides with potential use as ingredi-
ents for functional food systems.
Acknowledgments
This research forms part of Project 154307 Investigacin cien-
tca dirigida al desarrollo de derivados protenicos de Mucuna
pruriens con potencial actividad biolgica para la prevencin y/o
tratamiento de enfermedades crnicas asociadas al sobrepeso y la
obesidad, nanced by the Consejo Nacional de Ciencia y Tecnologa
(CONACYT).
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