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Research Article
Received: 22 March 2011 Revised: 28 November 2011 Accepted: 29 November 2011 Published online in Wiley Online Library: 13 January 2012

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5573

Hypocholesterolaemic and antioxidant

activities of chickpea (Cicer arietinum L.)
protein hydrolysates
Marı́a del Mar Yust,∗ Marı́a del Carmen Millán-Linares,
Juan Marı́a Alcaide-Hidalgo, Francisco Millán and Justo Pedroche

Abstract
BACKGROUND: Some dietary proteins possess biological properties which make them potential ingredients of functional or
health-promoting foods. Many of these properties are attributed to bioactive peptides that can be released by controlled
hydrolysis using exogenous proteases. The aim of this work was to test the improvement of hypocholesterolaemic and
antioxidant activities of chickpea protein isolate by means of hydrolysis with alcalase and flavourzyme.

RESULTS: All hydrolysates tested exhibited better hypocholesterolaemic activity when compared with chickpea protein isolate.
The highest cholesterol micellar solubility inhibition (50%) was found after 60 min of treatment with alcalase followed by
30 min of hydrolysis with flavourzyme. To test antioxidant activity of chickpea proteins three methods were used: β-carotene
bleaching method, reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect since antioxidant
activity of protein hydrolysates may not be attributed to a single mechanism. Chickpea hydrolysates showed better antioxidant
activity in all assays, especially reducing power and DPPH scavenging effect than chickpea protein isolate.

CONCLUSION: The results of this study showed the good potential of chickpea protein hydrolysates as bioactive ingredients.
The highest bioactive properties could be obtained by selecting the type of proteases and the hydrolysis time.

c 2012 Society of Chemical Industry

Keywords: chickpea; protein hydrolysates; hypocholesterolaemic activity; antioxidant activity

INTRODUCTION dant activities whereas other bioactive properties such as the

Nutritionally, proteins are a source of energy and amino acids,
which are essential for growth and maintenance. Functionally,
proteins contribute to the physicochemical and sensory properties
of various protein-rich foods. Furthermore, many dietary proteins
possess biological properties which make these components
potential ingredients of functional or health-promoting foods.
Many of these properties are attributed to biologically active
peptides encrypted in protein molecules that can be released
during the gastrointestinal digestion or by controlled hydrolytic
processes using exogenous proteases.1
Up to now, the main bioactive peptides described are from
animal sources, especially milk proteins. Pulse seed proteins
are less expensive and therefore offer a valuable alternative
for the production of food protein derived bioactive peptides.2
Among pulses, soy is by far the most studied. Recently, the
increased demand for plant-based proteins as food ingredients
has encouraged studies on proteins from soybean alternative
legumes such as chickpeas, common beans, peas, lentils and
lupins.3
In this sense, plant protein hydrolysates obtained by treatment
with proteases have been reported to carry specific bioactiv-
ities, and so constitute a group of under-exploited functional
food ingredients. Much research has focused on hydrolysates
1994
hypocholesterolaemic effect are less studied.
Regarding antioxidant activity of chickpea proteins, Arcan and
Yemenicioglu4 reported an increase in free radical scavenging
activity due to hydrolysis with pepsin whereas the antioxidant
potential in oil-in-water emulsion systems was almost unaffected
and the chelating capacity was reduced. Moreover, Li et al.5 found
that hydrolysis of 15% with alcalase resulted in an increase in free
radical scavenging activity, and inhibited linoleic acid autoxidation.
From this chickpea protein hydrolysate, a peptide with antioxidant
activity has been purified.6 However, the influence of the degree
of hydrolysis (DH) on antioxidant activity of chickpea hydrolysates
produced with alcalase has not been tested previously. Likewise,
DH has been reported to be a determinant factor in the generation
of peptides with biological activities (such as inhibition of
angiotensin converting enzyme-I and radical scavenging activities)
from plant proteins.7,8
With regard to hypocholesterolaemic activity, Pittaway et al.9
reported that the intake of chickpeas led to a decrease in serum
total cholesterol, although this effect was attributed to a higher
∗ Correspondence to: Marı́a del Mar Yust, Instituto de la Grasa-CSIC, Av. Padre
Garcı́a Tejero, 4, 41012-Seville, Spain. E-mail: mdmar@cica.es

with angiotensin-converting enzyme inhibitory and antioxi- Instituto de la Grasa-CSIC, Av. Padre Garcı́a Tejero, 4, 41012-Seville, Spain

J Sci Food Agric 2012; 92: 1994–2001 www.soci.org

c 2012 Society of Chemical Industry

Some biological properties of chickpea protein hydrolysates
intake of polyunsaturated fatty acids and dietary fibre. To our
knowledge, the hypocholesterolaemic effects of chickpea peptides
obtained after enzymatic hydrolysis have not been studied.
Since most bioactive peptides described at present are in the
size range of two to 20 amino acids and molecular masses of less
than 6000 Da,10 in this study we have tested hypocholesterolaemic
and antioxidant activities of chickpea hydrolysates ranging in DH
= 20–70% obtained by means of two commercially available
enzymes, alcalase and flavourzyme.
EXPERIMENTAL
Materials
Chickpea seeds were purchased from a local market. Alcalase
(2.4 L) and flavourzyme (1000 L) were a gift from Novozymes
(Bagsvaerd, Denmark). Cholesterol, trinitrobenzenesulfonic acid
(TNBS), butylated hydroxytoluene (BHT), and 2,2-diphenyl-1-
picrylhydrazyl (DPPH) were purchased from Sigma Chemical Co.
(St Louis, MO, USA). All other chemicals were of analytical grade.
Preparation of chickpea protein isolate
Chickpea protein isolate (CPI) was prepared according to Yust
et al.11 Briefly, chickpea defatted flour was suspended in water
and extracted at pH 10.5 for 1 h at room temperature. After
centrifuging, the supernatant was adjusted to the isoelectric point
(4.3) of chickpea proteins. The precipitate formed was recovered
by centrifuging, and spray dried.
Enzymatic hydrolysis of chickpea protein isolate
Hydrolysis of CPI was performed by sequential treatment with
alcalase (for 1 h) and flavourzyme (for 2 h) according to Clemente
et al.12 with slight modifications. The process was carried out in
a reactor with stirring and controlled pH and temperature. The
following hydrolysis parameters were used:
• alcalase hydrolysis: substrate concentration, 50 g L−1 ; enzyme
concentration, 4.6 mL L−1 ; temperature, 50 ◦ C; pH 8
• flavourzyme hydrolysis: substrate concentration, 50 g L−1 ; en-
zyme concentration, 1.85 mL L−1 ; temperature, 50 ◦ C; pH 7
During the course of the reaction, the pH was kept constant
by titration of the released protons with a NaOH solution in
appropriate concentration. Samples were withdrawn at different
times and enzymes were inactivated by heating at 85 ◦ C for 10 min.
Hydrolysates were centrifuged at 10 000 × g for 15 min to remove
insoluble material and freeze dried for further use.
Determination of the degree of hydrolysis
The DH, defined as the percentage of peptide bonds cleaved,
was measured by the TNBS method.13 The total number of
amino groups was determined in a sample that had been 100%
hydrolysed at 110 ◦ C for 24 h in 6 mol L−1 HCl.
Analytical methods
Protein and peptide amounts were determined by elemental
analysis, with a nitrogen-to-protein ratio of 6.25, using a LECO
CHNS-932 analyser (St Joseph, MI, USA). Total dietary fibre was
determined according to the procedure described by Lee et al.14
Ash content was determined by the direct ignition method (550 ◦ C
for 36 h). Polyphenols and soluble sugars were measured using
standard curves of chlorogenic acid15 and glucose,16 respectively.
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The quantification of amino acids was done according to Alaiz
et al.17 Tryptophan content was analysed according to the method
of Yust et al.18
Determination of hypocholesterolaemic activity
The in vitro micellar solubility of cholesterol in the presence
of chickpea protein hydrolysates (CPHs) was measured by the
method of Nagaoka et al.19 with some modifications. Micellar
solutions (10 mL) containing 10 mmol L−1 sodium taurocholate,
0.4 mmol L−1 cholesterol, 1 mmol L−1 oleic acid, 132 mmol L−1
NaCl, 15 mmol L−1 sodium phosphate (pH 7.4), and 2.5 mg mL−1
protein of tested samples were prepared by sonication. Then, the
mixture was incubated at 37 ◦ C for 24 h and ultracentrifuged at
140 000 × g for 60 min at 37 ◦ C. Cholesterol not incorporated
into micelles that remained in the supernatants after ultra-
centrifugation was extracted three times with chloroform, and
determined by gas chromatography. For this, a Hewlett-Packard
GC 5890 model series II, fitted with a flame ionization detec-
tor and a HP 3390A integrator (Palo Alto, CA, USA), was used.
Hydrogen at 12 psi column head pressure and 1 mL min−1 flow
rate was employed as the carrier gas. Nitrogen was used as an
auxiliary gas. Cholesterol was derivatised using a mixture of pyri-
dine–hexamethyldisilane–trimethylchlorosilane (9 : 3:1) at room
temperature for 15 min. Gas chromatography analyses were per-
formed using a HP-5 25 m × 0.32 mm × 0.17 µm capillary column.
Injector and detector temperature were maintained at 300 and
325 ◦ C, respectively. The oven temperature was maintained at
255 ◦ C.
Determination of antioxidant activity
β-Carotene bleaching method
The oxidative losses of β-carotene in a β-carotene–linoleic acid
emulsion were used to assess the antioxidant ability of CPI and
CPHs.20 Two milligrams of β-carotene were dissolved in 10 mL of
chloroform and 1 mL β-carotene solution was mixed with 20 mg
of linoleic acid and 200 mg of Tween 20. Then, chloroform was
removed in a rotary vacuum evaporator. Distilled water (50 mL)
enriched in oxygen was added and the resulting mixture was
vigorously stirred. An aliquot (2.5 mL) of the β-carotene–linoleic
acid emulsion was transferred to tubes containing 0.1 mL of each
sample (at 10 mg mL−1 protein), BHT (at 0.08 mg mL−1 ) or water
(control). An absorbance at 470 nm was immediately recorded
after adding the sample, which was regarded as t = 0 min. The
tubes were incubated at 50 ◦ C for 60 min and absorbance was
measured. Index protection (IP) was calculated according to the
equation IP = (A/A0 ) × 100, where A is absorbance at 60 min and
A0 is the absorbance at time zero.
Reducing power
The ability of the samples to reduce iron(III) was measured
according to Oyaizu.21 A 0.1 mL of sample (at concentration of
10 mg mL−1 protein) was added to 0.25 mL of 0.2 mol L−1 sodium
phosphate buffer pH 6.6 and 0.25 mL of 0.03 mol L−1 potassium
ferricyanide. The mixture was incubated at 50 ◦ C for 20 min. Then
0.25 mL of 0.6 mol L−1 trichloroacetic acid was added and the
mixture was centrifuged at 1300 × g for 10 min. Five hundred
microlitres of the upper layer were mixed with 0.5 mL of distilled
water and 0.1 mL of 3.7 mmol L−1 ferric chloride. After 10 min,
absorbance of the resulting solutions was measured at 700 nm.
Increased absorbance of the reaction mixture indicated increased
1995
reducing power. BHT at 0.08 mg mL−1 was used as standard.
wileyonlinelibrary.com/jsfa

www.soci.org
Figure 1. Time course of the hydrolysis of CPI by alcalase and flavourzyme.
DPPH radical-scavenging effect
The scavenging effect of the samples on DPPH free radical was
measured according to Wu et al.22 Briefly, 1.5 mL of each sample
(at concentration of 10 mg mL−1 protein) was added to 1.5 mL of
0.1 mmol L−1 DPPH in ethanol. The mixture was shaken and left
for 30 min at room temperature, and absorbance was measured
at 517 nm. The control was conducted in the same manner but
distilled water was used instead of sample. BHT at 0.08 mg mL−1
was used as positive standard. In its radical form, DPPH has an
absorption band at 517 nm which disappears upon reduction by an
anti-radical compound. Lower absorbance of the reaction mixture
represents higher DPPH scavenging activity. The scavenging effect
was expressed as [(Acontrol − Asample )/Acontrol ] × 100, where Asample
and Acontrol are the absorbances of the sample and control,
respectively.
Statistical analysis
Data were expressed as the mean ± standard deviation of three
independent determinations. Statistical analysis was performed
with the STATGRAPHICS Plus 5.0 program (Statistical Graphic,
Rockville, MD, USA). Data were analysed with one-way ANOVA
Tests and multiple range tests were conducted with Fisher’s least
significant difference procedure. Differences between the means
were considered to be significant when P < 0.05.
RESULTS AND DISCUSSION
Hydrolysis of chickpea protein isolate
Previous results indicated that the use of single enzymes, alcalase
or flavourzyme, led to the production of CPHs with DH <30%,
where peptides of 3–20 kDa are predominant.12 However, most
bioactive peptides described up to now have a molecular weight
below 1 kDa. For this reason, the extensive hydrolysis of CPI
was performed using both enzymes in a sequential way. First,
CPI was hydrolysed for 1 h with alcalase, which facilitated the
subsequent action of flavourzyme. DH evolution as time function
is showed in Fig. 1. The rate of hydrolysis was fast during the first
30 min of hydrolysis with alcalase and remained almost stable in
the following 30 min. The addition of flavourzyme led to a new
increase of DH up to 70% after 120 min of hydrolysis.
Second, hydrolysates obtained after 10, 20, 30, 40, 50 and 60 min
of hydrolysis with alcalase and after 60 min of hydrolysis with
alcalase followed by 15, 30, 45, 60, 75, 90 and 120 min of hydrolysis
1996
with flavourzyme were selected to study hypocholesterolaemic
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M del Mar Yust et al.
and antioxidant activities. These hydrolysates were named as
follows: 10A, 20A, 30A, 40A, 50A, 60A, A+15F, A+30F, A+45F,
A+60F, A+75F, A+90F and A+120F.
Hypocholesterolaemic activity
In the last decade, food proteins, peptides derived from their
hydrolysis and cholesterol absorption have received a great
deal of attention. In this sense, it has been shown that quality
and quantity of proteins in foods can alter serum cholesterol
concentration.23 – 26 Sugano et al.27 reported that a pepsin soybean
hydrolysate reduced serum cholesterol to a greater extent than
intact protein.
Cholesterol absorption by humans requires its solubilisation
in micelles formed by bile salts,28 and it has been reported
that peptides derived from digestion of food proteins may
decrease cholesterol solubility by competing with it for the
micellar composition.19 Therefore, studies on cholesterol micellar
solubility in presence of CPI and CPHs are a good test of their
hypocholesterolaemic activity. Results, expressed as percentage
of inhibition of cholesterol micellar concentration, are shown
in Fig. 2. CPI showed poor reduction of cholesterol micellar
solubility (only 4%), which is in agreement with results obtained
in our group for a Brassica carinata protein isolate1 but disagrees
with data obtained by Nagaoka et al.19 for soy protein isolates.
However, non-protein components, such as fructose, fibre and
saponins that may be present in protein isolates, seem to be
responsible of this effect in soy.29 All CPHs tested showed better
hypocholesterolaemic activity than CPI, with the exception of CPHs
10A and 20A, which were not significantly different compared
with CPI. Furthermore, the reduction of cholesterol micellar
solubility did not correlate with DH (r2 = 0.71). Although, initially,
cholesterol micellar concentration was reduced as DH increased,
after 40 min of hydrolysis with alcalase this effect disappeared. This
may be attributed to the destruction of hypocholesterolaemic
peptides and the generation of new ones, in such a way that
hypocholesterolaemic effect did not depend on DH but on the
generation of specific peptides.
The hydrolysate with the highest hypocholesterolaemic effect
was obtained after 60 min of hydrolysis with alcalase followed
by 30 min of hydrolysis with flavourzyme (although there were
no significant differences with respect to A+90F and A+120F),
which reduced cholesterol micellar solubility by 50%. This result is
especially important if we compare it with data reported by other
authors. For instance, Nagaoka et al.19 obtained a 37% reduction
in cholesterol micellar solubility using concentrations of 10 mg
protein mL−1 (four times higher than the one employed in this
study) from a bovine whey β-lactoglobulin tryptic hydrolysate,
and 68% reduction with soybean peptic hydrolysates using
5 mg protein mL−1 . Zhong et al.30 obtained 48.6% reduction in
cholesterol micellar solubility using 5 mg mL−1 peptides from a
soy protein hydrolysate obtained with alcalase. Results obtained
with CPHs generally confirm these data and are similar to
hypocholesterolaemic effect reported by Pedroche et al.1 with
B. carinata protein hydrolysates.
The main difference between the composition of the CPI and
CPH A+30F is ash content (Table 1), higher in the hydrolysate,
which is a consequence of the addition of NaOH to keep the pH
constant during hydrolysis. Since neither polyphenols nor sugars
were detected in CPH A+30F, the hypocholesterolaemic effect
may be attributed to the protein and peptides. Additionally, amino
acid composition of CPI and CPH A+30F did not show significant
differences (Table 2), so the hypocholesterolaemic activity may be
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Some biological properties of chickpea protein hydrolysates www.soci.org

Figure 2. Hypocholesterolaemic activity of CPI and CPHs obtained by sequential hydrolysis with alcalase and flavourzyme. 10A, 20A, 30A, 40A, 50A,
60A: chickpea protein hydrolysates obtained after 10, 20, 30, 40, 50 and 60 min of hydrolysis with alcalase, respectively. A+15F, A+30F, A+45F, A+60F,
A+75F, A+90F, A+120F: Chickpea protein hydrolysates obtained after 60 min of hydrolysis with alcalase followed by 15, 30, 45, 60, 90 and 120 min
of hydrolysis with flavourzyme, respectively. BHT concentration: 0.08 mg mL−1 . CPI and CPHs concentration: 10 mg mL−1 . Data represent the mean ±
standard deviation of three determinations. Bars with a different letter are significantly different (P < 0.05).

Table 1. Composition of CPI and the CPHs with the highest hypocholesterolaemic activity and with the highest antioxidant activities

Component CPI CPH 30A CPH A+30F CPH A+90F CPH A+120F

Protein 924.5 ± 9.8 878.1 ± 7.0 840.5 ± 9.9 845.6 ± 4.7 835.0 ± 2.2
Total fibre 41.6 ± 4.0 9.2 ± 0.9 9.2 ± 0.3 12.0 ± 0.7 11.0 ± 0.4
Ash 24.0 ± 0.1 118.1 ± 1.7 147.0 ± 1.9 148.4 ± 3.4 150.3 ± 4.1
Polyphenols 0.2 ± 0.02 ND ND ND ND
Soluble sugars 4.1 ± 0.3 1.1 ± 0.1 ND ND ND

Results are expressed as g kg−1 of dry matter.

CPH 30A is CPH obtained after 30 min oh hydrolysis with Alcalase.
CPH A+30F, CPH A+90 F, and CPH A+120F are CPHs obtained after 60 min of hydrolysis with Alcalase followed by 30, 90, and 120 min of hydrolysis
with Flavourzyme, respectively.
Values represent mean ± standard deviation (n = 3).
ND, not detected.

due to the release of specific bioactive peptide from intact proteins
after enzymatic digestion.30
Antioxidant activity
The antioxidant activity of protein hydrolysates may not be
attributed to a single mechanism. Therefore, different assays were
performed to study the antioxidant properties of CPHs.31
In all of them, BHT was used as positive control and water
as a negative control. CPI and CPHs were tested at a higher
concentration than that of BHT, because antioxidant peptides
may be in low concentrations among the wide pool of peptides
that composed the protein hydrolysates. Moreover, it has been
reported that many natural antioxidants are less potent than the
synthetic ones, but they can be used at higher concentrations
due to the very restrictive toxicological parameters of synthetic
antioxidants. In addition, the incorporation of protein hydrolysates
into foods could confer desirable nutritional and functional
β-Carotene bleaching method
Decoloration of β-carotene due to oxidation products of linoleic
acid in presence of CPI and CPHs was studied. This assay is widely
used to measure the antioxidant activity of bioactive compounds
because β-carotene is extremely susceptible to free radical
mediated oxidation of linoleic acid.33 CPI and CPHs antioxidant
activities after 60 min of reaction, expressed as protection index,
are shown in Fig. 3. CPI presented 26% protection index. In
early stages of hydrolysis, the antioxidant effect increased with
increasing time, achieving maximum activity after 30 min of
hydrolysis with alcalase; this hydrolysate was approximately two-
fold more antioxidant than CPI. From this time on, the antioxidant
effect decreased as the DH increased, especially after flavourzyme
was added to the reaction media. It has been reported that the
increase in antioxidant activity of proteins after hydrolysis largely
depends on protease specificity, degree of hydrolysis and nature
of released peptides.34 At the end of the process, hydrolysates
1997

properties.32 showed almost the same or even lower protection index than

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Table 2. Amino acid composition of CPI and the CPHs with the highest hypocholesterolaemic activity

Amino acid CPI CPH 30A CPH A+30F CPH A+90F CPH A+120F FAOa

Asp+Asn 132.5 ± 5.3 129.7 ± 1.5 135.9 ± 3.4 130.8 ± 0.7 128.1 ± 3.0 –
Glu+Gln 170.6 ± 6.5 177.0 ± 1.3 174.8 ± 4.6 169.9 ± 1.6 175.7 ± 5.0 –
Ser 59.8 ± 1.9 61.3 ± 0.4 60.9 ± 1.6 61.8 ± 0.6 59.7 ± 1.0 –
His 27.7 ± 1.0 26.9 ± 0.2 26.6 ± 0.6 27.6 ± 0.2 26.4 ± 0.1 19
Gly 39.0 ± 0.9 41.0 ± 0.8 40.5 ± 1.1 42.0 ± 0.1 41.4 ± 2.0 –
Thr 45.4 ± 1.0 43.9 ± 0.7 42.3 ± 3.0 45.9 ± 1.0 44.8 ± 1.1 34
Arg 91.6 ± 0.8 91.1 ± 0.8 88.5 ± 2.2 89.8 ± 1.3 93.1 ± 1.2 –
Ala 48.5 ± 2.0 47.6 ± 0.5 46.2 ± 1.2 45.8 ± 1.1 46.9 ± 0.9 –
Pro 48.1 ± 6.3 42.3 ± 2.0 43.5 ± 3.5 45.2 ± 1.5 43.2 ± 0.6 –
Tyr 23.9 ± 0.4 25.1 ± 0.8 26.8 ± 0.6 23.0 ± 0.4 25.7 ± 0.7 –
Val 42.5 ± 2.1 40.3 ± 1.5 44.6 ± 1.1 44.2 ± 0.6 43.8 ± 0.2 35
Met 2.4 ± 0.2 1.9 ± 0.1 2.3 ± 0.2 2.8 ± 1.3 2.0 ± 0.2 25b
Cys 9.3 ± 0.9 10.0 ± 1.6 8.5 ± 0.2 9.5 ± 0.2 8.8 ± 1.8 –
Ile 33.8 ± 1.2 33.5 ± 1.5 35.9 ± 0.9 36.2 ± 0.2 35.4 ± 0.6 28
Leu 89.8 ± 2.7 90.6 ± 0.5 85.1 ± 2.0 87.5 ± 0.4 84.9 ± 0.2 66
Phe 64.4 ± 0.9 63.9 ± 0.6 63.5 ± 1.4 62.7 ± 0.1 65.6 ± 0.2 63c
Lys 64.2 ± 2.6 66.7 ± 1.6 67.2 ± 1.7 67.7 ± 0.4 65.4 ± 0.1 58
Trp 6.6 ± 0.3 7.0 ± 0.3 7.0 ± 0.2 7.5 ± 0.4 7.3 ± 0.1 –

Results are expressed as g kg−1 protein.

CPH 30A is the CPH obtained after 30 min of hydrolysis with Alcalase.
CPH A+30F, CPH A+90 F and CPH A+120F are the CPHs obtained after 60 min of hydrolysis with Alcalase followed by 30, 90 and 120 min of hydrolysis
with Flavourzyme, respectively.
Values represent mean ± standard deviation (n = 3).
a FAO/WHO/ONU. Energy and protein requirements, 1985.
b Methionine + cysteine.
c Phenylalanine + tyrosine.

CPI. This effect may be attributed to the hydrolysis of antioxidant
peptides generated in the first stage.
Reducing power
The reducing power assay is often used to evaluate the ability of
antioxidant proteins and peptides to donate electrons. Different
studies have reported that there is a direct correlation between
antioxidant activities and reducing power of certain bioactive
compounds.35 In this assay, the ability of CPHs to reduce Fe3+
to Fe2+ was determined. The presence of antioxidants in tested
samples results in the reduction of Fe3+ /ferricyanide complex to
ferrous form.
All CPHs showed better reducing power activity than CPI
(Fig. 4). The use of alcalase produced hydrolysates with similar
values of reducing power, independently of hydrolysis time, and
those were, on average, 2.16-fold higher than CPI. Regarding
hydrolysates obtained after hydrolysis with flavourzyme, there is
no correlation between this activity and DH, which may attributed
to the destruction of reductant peptides and the generation of
new ones throughout the hydrolytic process. The best result was
obtained in hydrolysate A+90F, whose reducing power was 2.53
times higher than CPI.
DPPH radical-scavenging activity
The DPPH radicals have been widely used to investigate the
scavenging activity of some natural compounds. DPPH is a stable
free radical that shows maximum absorbance at 517 nm. When
DPPH radical encounters a proton-donating substrate such as an
1998
reduced. Thus, the decrease in absorbance is taken as a measure
for DPPH-scavenging activity.
As shown in Fig. 5, all samples tested showed DPPH radical
scavenging activity. Furthermore, all hydrolysates had higher
activity than CPI, so it may be concluded that hydrolysis generated
peptides that could react with free radicals to convert them to more
stable products and complete the radical chain reaction. Hydrolysis
with alcalase increased the DPPH scavenging effect up to 50%.
The subsequent hydrolysis with flavourzyme increased the DPPH
scavenging activity even more, obtaining values of approximately
77% for A+45F, A+75F and A+120F. If we compare these
hydrolysates with CPI, the free radical scavenging effect has been
increased 2.3-fold. These data are lower than those reported by
Arcan and Yemenicioglu,4 who found that chickpea hydrolysates
produced with pepsin increased free radical scavenging activity
up to three-fold, although they used ABTS radical to test this effect.
On the other hand, the second step of hydrolysis with
flavourzyme seems to be the determinant in the release of
peptides with DPPH scavenging activity. Our results agree with
those reported by Je et al.36 in the hydrolysis of tuna liver using
a two-step enzymatic process, where flavourzyme is employed in
the second phase. These authors suggested that this effect may be
due to the different enzymatic specificities and the lower molecular
weight of hydrolysates obtained in the second hydrolysis, which
has been reported to have higher scavenging activity than high
molecular weight hydrolysates.22,37
Considering the three studied methods, hydrolysis of chickpea
proteins by the sequential action of alcalase and flavourzyme led to
an increase in antioxidant activity, although values are far from the
ones corresponding to BHT. However, the most active antioxidant,

antioxidant, the radicals are scavenged and the absorbance is CPH, depends on the specific assay. So, the best CPH in the β-

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Figure 3. Protection index of CPI and CPHs obtained by sequential hydrolysis with alcalase and flavourzyme. 10A, 20A, 30A, 40A, 50A, 60A: CPHs obtained
after 10, 20, 30, 40, 50 and 60 min of hydrolysis with alcalase, respectively. A+15F, A+30F, A+45F, A+60F, A+75F, A+90F, A+120F: CPHs obtained
after 60 min of hydrolysis with alcalase followed by 15, 30, 45, 60, 90 and 120 min of hydrolysis with flavourzyme, respectively. BHT concentration:
0.08 mg mL−1 . CPI and CPHs concentration: 10 mg mL−1 . Data represent the mean ± standard deviation of three determinations. Bars with a different
letter are significantly different (P < 0.05).

Figure 4. Reducing power of CPI and CPHs. 10A, 20A, 30A, 40A, 50A, 60A: CPHs obtained after 10, 20, 30, 40, 50 and 60 min of hydrolysis with alcalase,
respectively. A+15F, A+30F, A+45F, A+60F, A+75F, A+90F, A+120F: CPHs obtained after 60 min of hydrolysis with alcalase followed by 15, 30, 45, 60,
90 and 120 min of hydrolysis with flavourzyme, respectively. BHT concentration: 0.08 mg mL−1 . CPI and CPHs concentration: 10 mg mL−1 . Data represent
the mean ± standard deviation of three determinations. Bars with a different letter are significantly different (P < 0.05).

carotene bleaching method is 30A, whereas in reducing power it is
A+90F and in the DPPH assay it is A+120F. It seems that peptides
with different antioxidant mechanisms are generated throughout
the hydrolytic process, which agrees with results reported by Arcan
and Yemenicioglu4 when chickpea proteins were hydrolysed with
pepsin.
If we compare the composition of those three CPHs with
CPI (Table 1) the main differences are ash contents, which are
mentioned before, this effect is due to the addition of NaOH to
keep the pH constant during hydrolysis. With regards to amino acid
composition, no significant differences were observed between
CPI and the CPHs (Table 2). Similar results have been reported
by Sakanaka et al.,38 who obtained hydrolysates from casein with
higher antioxidant activity than the original casein but with the
same amino acid composition. In this sense, the increase of
antioxidant activity may be attributed to the release of specific
1999

higher as long as the hydrolysis time is increased. As we have amino acid sequences from intact protein.

J Sci Food Agric 2012; 92: 1994–2001

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Figure 5. DPPH radical-scavenging activity of CPI and CPHs. 10A, 20A, 30A, 40A, 50A, 60A: CPHs obtained after 10, 20, 30, 40, 50 and 60 min of hydrolysis
with alcalase, respectively. A+15F, A+30F, A+45F, A+60F, A+75F, A+90F, A+120F: CPHs obtained after 60 min of hydrolysis with alcalase followed by
15, 30, 45, 60, 90 and 120 min of hydrolysis with flavourzyme, respectively. BHT concentration: 0.08 mg mL−1 . CPI and CPHs concentration: 10 mg mL−1 .
Data represent the means ± standard deviation of three determinations. Bars with a different letter are significantly different (P < 0.05).
CONCLUSIONS
Chickpea hydrolysates obtained by action of commercial proteases
presented higher hypocholesterolaemic and antioxidant activity
than intact proteins. The hydrolysates with highest bioactivities
could be obtained by selecting the type of proteases and
the time of hydrolysis. Hypercholesterolaemia and oxidative
stress, together with hypertension, are the main factors in the
development of several pathologies, especially all those related
to heart and cardiovascular diseases. Results of our study show
the potential use of chickpea hydrolysates in the developing
of functional foods for the treatment of cardiovascular and
neurological pathologies. Additionally, the use of chickpea protein
hydrolysates as ingredients may reduce the need of adding
synthetic antioxidants to preserve food from rancidity. However,
further work should be done to isolate and identify the specific
peptides that are responsible for those beneficial effects.
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