Eur Food Res Technol (2010) 231:13–19
DOI 10.1007/s00217-010-1235-5
ORIGINAL PAPER
Production and characterization of angiotensin converting
enzyme (ACE) inhibitory peptides from apricot
(Prunus armeniaca L.) kernel protein hydrolysate
Zhenbao Zhu · Nongxue Qiu · Jianhua Yi
Received: 24 November 2009 / Revised: 26 January 2010 / Accepted: 3 February 2010 / Published online: 6 March 2010
© Springer-Verlag 2010
Abstract Six diVerent proteases (Flavourzyme®, Neutr-
ase®, Protamex®, Alcalase® 2.4L, Proleather® FG-F, and
papain) were employed to hydrolyze apricot kernel protein
(AKP). Alcalase® is an inexpensive and non-speciWc prote-
ase that has been shown to be useful for the generation of
bioactive peptides from AKP. Alcalase® 2.4L was selected
for further study on enzymatic preparation of ACE inhibi-
tory peptide from AKP. After 60-min hydrolysis, the high-
est ACE inhibition was 82 § 0.14%. Results of molecular
weight distribution revealed that most of ACE inhibition
activity was probably attributed to low-molecular weight
peptide fraction ranging from 200 to 900 Da. UltraWltration
on membranes with several molecular weight cutoVs
(MWCFs) demonstrated that most of the ACE inhibitory
activity was due to peptides with a less than 1,000 Da
molecular weight: the IC50 value of the 1-kDa ultraWltrate
was 0.15 § 0.007 mg mL¡1, while it was 0.378 §
0.015 mg mL¡1 before ultraWltration. Additionally, further
separation and puriWcation of the ACE inhibitory peptides
were carried out using gel Wltration and C18 RP-HPLC. The
result of research can be used to optimize AKP enzymatic
Z. Zhu · N. Qiu (&)
College of Life Science, Shaanxi Normal University,
Xi’an 710062, China
e-mail: nongxueq@snnu.edu.cn; qiunx@163.com
Z. Zhu · J. Yi
College of Life Science and Engineering,
Shaanxi University of Science and Technology,
Xi’an 710021, China
N. Qiu
College of Food Engineering and Nutritional Science,
Shaanxi Normal University, Xi’an 710062, China
hydrolysis for producing ACE inhibitory peptides which
could be used for food industry and nutraceuticals.
Keywords Apricot kernel protein hydrolysate ·
Angiotensin l-converting enzyme (ACE) · Bioactive
peptides · UltraWltration · Prunus armeniaca L
Introduction
Apricot (Prunus armeniaca L.) is a popular stone fruit
grown in many countries, such as China, Turkey, and
Egypt. Abundant amounts of apricots are consumed yearly,
either as fresh fruit or processed into apricot juice, nectar,
jam, or dried fruit during the summer growing season.
A fairly large number of apricot seeds are produced as a
by-product of the processing industry. Apricot kernels
contain a high percentage of oil (50%) that can be used for
human nutrition [1], cosmetics, and medicine. Because of
their appreciable protein content, apricot kernels are a good
source of food [2, 3]. Unprocessed apricot kernels can not
be used directly as a food source due to their high levels of
amygdalin. However, it was previously reported that detox-
iWed apricot kernels were nutritionally balanced and com-
pletely safe to incorporate in food products [4].
Hypertension is a signiWcant health problem worldwide.
It is one of the important risk factors associated with devel-
oping disorders such as stroke, coronary artery disease,
myocardial infarction, heart failure, abnormal renal func-
tion leading to renal failure, and many other complications
associated with structural damage to the cardiovascular system
[5]. Angiotensin converting enzyme (ACE; dipeptidylcarboxy-
peptidase; EC 3.4.15.1) plays a central role in the regula-
tion of blood pressure by virtue of renin-angiotensin system
[6]. ACE converts the inactive decapeptide (angiotensin I)
123
14
to the potent vasopressor octapeptide (angiotensin II) and
also inactivates the antihypertensive vasodilator bradyki-
nin. Since the discovery of ACE inhibitors in snake venom,
many synthetic ACE inhibitors such as captopril, lisinopril,
and enalapril are currently used as pharmaceuticals to treat
hypertension [7]. However, these synthetic drugs are
known to have certain side eVects such as cough, taste dis-
turbance, and skin rashes. In contrast, the food-protein-
derived ACE inhibitory peptides have not exhibited these
side eVects yet [8].
In recent years, ACE inhibitory peptides have been
derived from numerous food sources including beef meat
hydrolysates [9], fermented oyster sauce [10], porcine
hemoglobin [11], ovalbumin [12], and fermented milk [13].
It should be noted that several ACE inhibitory peptides were
isolated from plant protein hydrolysates such as soy bean
[14, 15], defatted canola meal [16], algae proteins hydroly-
sates [17, 18], and mung bean protein [19]. However, to the
best of our knowledge, there is no information to produce
ACE inhibitory peptides from apricot kernel protein.
Because patients with hypertension often need life-long
medical treatment, interest has been focused on the isola-
tion and identiWcation of ACE inhibitory peptides which
may be obtained from new and varied food sources. There-
fore, as a series of research on preparation of bioactive pep-
tide from Chinese bitter apricot kernel protein, the objective
of this work was to obtain ACE inhibitory peptides during
hydrolysis of apricot kernel protein using diVerent protease
and to purify ACE inhibitory peptides by ultraWltration.
Also, the apricot kernel protein Alcalase® hydrolysate was
characterized by gel Wltration and C18 RP-HPLC. In addi-
tion, we have isolated a pentapeptide with 688-Da molecu-
lar weight that showed the highest ACE inhibitory activity
after fractionation of this hydrolysate using consecutive
chromatographic techniques (manuscript in prepara-
tion).We show that an underutilized by-product such as
apricot kernel may be useful in human food nutrition as a
source of bioactive peptides with potential antihypertensive
properties.
Materials and methods
Materials
The apricot kernel protein was prepared in our laboratory
by alkali extraction and acid precipitation method. Alca-
lase®2.4L, Flavourzyme®, Neutrase®, and Protamex® were
obtained from Novozymes (Bagsvaerd, Denmark). Papain,
pepsin (porcine stomach mucosa), trypsin (bovine pan-
creas), and chymotrypsin (bovine pancreas) were purchased
from a local chemical reagent shop. Proleather ®FG-F was
a gift from Amano Enzyme Inc. (Nagoya, Japan). ACE
123
Eur Food Res Technol (2010) 231:13–19
(from rabbit lung), HHL (Hippuryl-L-histidyl-L-leucine),
HA (hippuric acid), and captopril were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Sephadex G15
was obtained from GE healthcare Inc. (GE, Beijing, China).
Other reagents used were of analytical grade.
Enzymatic hydrolysis of apricot kernel protein
To produce ACE inhibitory peptides from apricot kernel
protein, enzymatic hydrolysis was performed using various
enzymes (Alcalase®, Flavourzyme®, Neutrase®, Prota-
mex®, Proleather® FG-F, and papain) under optimal condi-
tions (Table 1) at an enzyme/substrate ratio of 2:100(w/w).
Then, 2%(w/w) AKP solutions and enzymes were mixed.
The degree of hydrolysis (DH) was determined according
to the pH–stat method as described previously [20]. After
maintaining the pH constant with 1.0 mol L¡1 NaOH dur-
ing hydrolysis, the enzyme was inactivated at 90 °C for
10 min, and the hydrolysate solution was cooled down to
room temperature, neutralized, and centrifuged at 10,000g
for 30 min. The supernatants were lyophilized and stored at
¡20 °C until further analysis within a month.
The ACE inhibitory activity of hydrolysates prepared
with Proleather® FG-F and Alcalase® 2.4L during
in vitro gastrointestinal digestion
In order to check the impact of gastrointestinal proteases on
the ACE inhibitory activity of hydrolysates obtained with
Proleather® FG-F and Alcalase® 2.4L, subsequent hydroly-
sis with pepsin, trypsin, and
-chymotrypsin was conducted
to simulate the human gastrointestinal digestion process.
BrieXy, a 100-mL volume of 10 mg mL¡1 enzymatic
hydrolysate(after 240-min hydrolysis) pH was adjusted to
2.0 with 4 mol L¡1 HCl, adding pepsin ([E]/[S] = 1:200),
and incubating for 2 h at 37 °C. Then, trypsin and
-chymo-
trypsin (1:1, [E]/[S] = 1:200) at pH 6.5 (with 5 mol L¡1
NaOH) and incubation for 3 h at 37 °C simulated the small
intestine phase. The enzyme was inactivated at 90 °C for
10 min, and the hydrolysate solution was cooled down to
room temperature, neutralized, and centrifuged at 12,000g
for 30 min. The supernatants were lyophilized and stored at
¡20 °C for assay of ACE inhibitory activity.
Assay of ACE inhibitory activity
The ACE inhibitory activity was measured by the direct
HPLC injection method as reported [21] with slight modiW-
cations. BrieXy, a total volume of 70
L (all prepared with
50 mmol L¡1 sodium borate buVer containing 300 mmol
L¡1 NaCl at pH 8.3) consisting of 50
L of 6.15 mmol L¡1
HHL, 2mU of ACE, and a solution of the freeze-dried pro-
tein hydrolysate (2.5 mg mL¡1, 10
L) were used. After a
Eur Food Res Technol (2010) 231:13–19
10-min incubation of HHL and enzymatic hydrolysate, the
ACE was added to initiate reaction at 37 °C in 30 min.
Finally, the reaction was terminated by the addition of
85
L 1 mol L¡1 HCl, and 10
L of this solution was
injected into a RP-HPLC C18 column (Diamonsil™, Beijing,
China) for HA separation and quantiWcation. The operation
was performed on a Dionex HPLC system equipped with a
P680 HPLC pump, an UVD170U detector (Dionex, Sunny-
vale, USA), and an Autoscience™ AT-330 column heater.
The mobile phase was: (a) 25% acetonitrile and (b) 0.1%
triXuoroacetic acid (TFA) in Milli-Q water with a Xow rate
of 1.0 mL min¡1. The column was eluted with an isocratic
elution. The eZuent was monitored with an ultraviolet
detector at 228 nm. External HA standard samples were
prepared fresh daily and used for calculation of HA formed
in test reactions. The IC50 value, deWned as the concentra-
tion of the peptide inhibiting 50% of the ACE activity, was
determined by measuring the ACE inhibitory activity ver-
sus peptide contents of each sample after the regression
analysis at least Wve separate determinations. Also, the IC50
value of captopril (a well-known synthetic ACE inhibitor)
was determined. The peptide contents were assayed by the
Bradford method [22].
Determination of molecular weight distribution for apricot
kernel protein hydrolysate (AKPH)
The apricot kernel protein hydrolysate (AKPH) obtained
with Alcalase® was analyzed for molecular weight distribu-
tion using a Waters™ 600E Advanced Protein PuriWcation
System (Waters Corporation, Milford, MA, USA). The
hydrolysates were loaded onto a TSK G2000 SWXL gel
Wltration column (7.8 i.d. £ 300 mm, Tosoh, Tokyo,
Japan), eluted with a 55%(v/v) water–45% (v/v) acetoni-
trile mixture containing 0.1% (v/v) TFA at a Xow rate of
0.5 mL min¡1, and monitored at 220 nm. A molecular
weight calibration curve was obtained from the following
Table 1 ACE inhibition activity, degree of hydrolysis, and optimum hydrolysis conditions of individual enzymes
Enzyme EC number Optimum conditions
pH T (°C)
Protamex® 3.4.24.28 7.0 50
®
Alcalase 2.4L 3.4.21.62 8.0 55
Proleather® FG-F 3.4.21.62 10 60
Neutrase® 3.4.24.28 7.0 50
Flavourzyme® 3.4.11.1 7.0 50
Papain 3.4.22.2 7.0 55
a ¡1
Concentration of enzyme hydrolysate is 2.5 mg mL
b
Degree of hydrolysis was determined at 240 min
15
standards from Sigma: cytochrome C (12,500 Da), aproti-
nin (6,500 Da), bacitracin (1,450 Da), tetrapeptide GGYR
(451 Da), and tripeptide GGG (189 Da). Results were
obtained and processed using Millennium32 Version 3.05
software (Waters Corporation, Milford, MA, USA).
EVect of ultraWltration on AKP hydrolysates ACE
inhibitory activity
AKPHs generated by Alcalase®2.4L were subjected to
ultraWltration with the Millipore system (Millipore, CA,
USA) using membranes with either 5- or 1-kDa molecular
weight cutoVs(MWCOs). The hydrolysates were thus
divided into three fractions by ultraWltration, namely
AKPH I (>5 kDa), AKPHII (1–5 kDa), and AKPHIII
(<1 kDa). Each fraction was collected and lyophilized to
determine its protein concentration and IC50 for ACE
inhibitory activity.
Fractionation of AKPHIII (<1 kDa) by gel Wltration
chromatography
The AKPHIII (<1 kDa) was subjected to gel Wltration chro-
matography. The peptide solution was loaded onto a Sepha-
dex G15 column (1.6 £ 80 cm; Pharmacia Co., Uppsala,
Sweden) pre-equilibrated with water and eluted at a Xow
rate of 0.5 mL min¡1. Samples were eluted by 0.1% TFA in
water, and 3-mL fractions were collected at a Xow rate of
0.5 mL min¡1. The eZuent was monitored with an ultravio-
let detector at 280 nm. Fractions were pooled and lyophi-
lized for RP-HPLC.
RP-HPLC chromatograms of AKPH
RP-HPLC is widely utilized to generate a peptide map from
digested protein. The peptide mixture of AKPHIII (<1 kDa)
was separated by RP-HPLC on a symmetry C18 column
ACE inhibition Degree of
activity (%)a hydrolysis (DH/%)b
76 § 0.09 7.5 § 0.49
74 § 0.62 18 § 0.14
67 § 0.50 18 § 0.75
69 § 0.08 5 § 0.23
4 § 0.54 5 § 0.69
74 § 0.74 3 § 0.23
123
16
(4.6 £ 250 mm, 5
m). Also, two fractions from Sephadex
G15 were puriWed using the same conditions. The mobile
phase was delivered by Dionex P680 HPLC pump (Dionex,
Sunnyvale, USA). Separation was made under linear gradi-
ent elution conditions using acetonitrile as the organic mod-
iWer and TFA as the volatile buVer. The mobile phase was:
(A) 0.1% triXuoroacetic acid (TFA) + Milli-Q water (B)
0.07% TFA + acetonitrile with a Xow rate of 1.0 mL min¡1.
The chromatographic column was conditioned with 90% of
eluent A, after which 20
L of the sample was applied on
the C18 column and eluted with the following eluent B con-
centrations: 0–30 min, 10–25%; 30–40 min, 25–10%; and
40–50 min, 10%. The UV absorbance of the eluent was
monitored at 214 nm.
Statistical analysis
Results were presented as means of experiments done in
triplicate § standard deviation (SD). The Student’s t-test
was used to determine the level of signiWcance. A p value
of less than 0.05 was considered as signiWcant.
Results and discussion
EVect of diVerent protease hydrolyses on ACE inhibitory
activity
It has been reported that the IC50 values for ACE inhibitory
activity of 48 sea protein hydrolysates were aVected by
both the marine protein resources and the selected proteases
[23]. In this study, apricot kernel protein (AKP) was hydro-
lyzed by Alcalase®, Flavourzyme®, Neutrase®, Protamex®,
Proleather®FG-F, and papain for 0.5–4 h, respectively.
Table 1 shows the comparison of ACE inhibitory activities
and degrees of hydrolysis (DH), respectively, within six
enzyme hydrolysates as a function of hydrolysis time.
Hydrolysates prepared with all enzymes, except Flavour-
zyme®, exhibited higher ACE inhibitory activities than
native AKP and varied from 67 § 0.5% to 76 § 0.09%
inhibition. At the same time, AKP showed no ACE inhibi-
tory activity. The results indicate that enzymatic hydrolysis
was required to release ACE inhibitory peptides from the
inactive form of intact AKP. However, the DH of six
enzyme hydrolysates showed signiWcant diVerence (Fig. 1;
Table 1). Alcalase® and Proleather® FG-F showed over
18% of DH, while papain showed 3 § 0.23% of DH. Inter-
estingly, there appeared to be no direct relationship
between ACE inhibitory activities and the DH of the
hydrolysates. For example, the papain hydrolysates inhib-
ited 74 § 0.74% ACE activity. On the other hand, the Fla-
vourzyme® hydrolysates inhibited only 4 § 0.54% ACE
activity with a 5 § 0.69% DH.
123
Eur Food Res Technol (2010) 231:13–19
20
15 Protamex
ProleatherFG-F
Alcalase2.4L
Neutrase
DH(%)
Flavourzyme
10
Papain
5
0
0 30 60 90 120 150 180 210 240
Hydrolysis time(min)
Fig. 1 Comparison of degree of hydrolysis (DH) within six enzyme
hydrolysates as a function of hydrolysis time. Results are mean values
of at least two replicates
As can be seen in Table 1, among the six enzymes, Alca-
lase® hydrolysate inhibited 74 § 0.62% ACE activity and
for a 18 § 0.14% DH. Therefore, Alcalase® was selected
for further study on enzymatic preparation of ACE inhibi-
tory peptides from AKP considering both ACE inhibitory
activity and DH. Additionally, Alcalase® is an alkaline pro-
tease for widely industrial use produced from Bacillus
licheniformis, and the main enzyme component is subtilisin
Carlsberg (a serine protease). In recent years, Alcalase® has
been extensively studied to hydrolyze various plant pro-
teins such as soy protein isolates [15], defatted canola meal
[16], mung bean protein [19], and chickpea legumin [24],
to produce ACE inhibitors.
It is well known that not all potent peptide inhibitors of
ACE in vitro may necessarily be antihypertensive in vivo.
Some peptides can be susceptible to degradation or modiW-
cation in the gastrointestinal digestion process. It is impor-
tant to note that antihypertensive peptides are generally
resistant to degradation by digestive enzymes. The stability
of each hydrolysate prepared with Proleather® FG-F and
Alcalase® 2.4L against gastrointestinal proteinases in vitro
was examined in order to predict their antihypertensive
eVects in vivo (Table 2). The ACE inhibitory activities of
Proleather® FG-F and Alcalase® 2.4L were almost preserved
after the digestion. The result indicated that Alcalase®
2.4L hydrolysates can be used to purify antihypertensive
peptides.
EVect of hydrolysis time on ACE inhibitory activity
of apricot kernel protein Alcalase® hydrolysates
The eVect of hydrolysis time on the ACE inhibitory activity
was further evaluated under the optimum hydrolysis condi-
tions for Alcalase® (Fig. 2). The ACE inhibitory activity of
Eur Food Res Technol (2010) 231:13–19 17

Table 2 Digestive stability of the ACE inhibitory activity from 1.00

254
Proleather® FG-F and Alcalase® 2.4 L hydrolysates

301
0.80
Before digestion (%)a After digestion (%)

534
0.60

636
369
AU
Alcalase® 2.4L 75 § 0.81 73.49 § 0.75
0.40

871
Proleather® FG-F 67.1 § 0.48 68 § 0.27

1026
0.20

2631
Each hydrolysate was successively treated with pepsin, trypsin, and

4524
chymotrypsin as described in the materials and methods section
a
0.00
Concentration of enzyme hydrolysate is 2.5 mg mL¡1
5.00 10.00 15.00 20.00 25.00 30.00
Minutes
a a a
b a Fig. 3 Molecular weight distribution of AKPH fraction having the
80 b
highest ACE inhibition activity (i.e., after 60-min hydrolysis by Alca-
lase®). GPC/HPLC conditions: column, TSK Gel2000 SWXL (7.8 mm
i.d. £ 300 mm); mobile phase, acetonitrile/water/triXuoroacetic acid
ACE inhibition activity(%)

60 (45:55:0.1 v/v/v); Xow rate, 0.5 mL min¡1; and detection, absorbance

at 220 nm. Values above peaks indicate molecular weights

40
20
0
0 30 60 90 120
Hydrolysis time(min)
Fig. 2 EVect of hydrolysis time on ACE inhibition activity of apricot
kernel protein Alcalase®2.4L hydrolysates. The values were repre-
sented as the mean of the triplicate § SD. Values with diVerent letters
are signiWcantly diVerent (p < 0.05)
hydrolysates increased up to 60-min hydrolysis. As can be
seen in Fig. 2, the 60-min Alcalase® protein hydrolysate
showed the highest ACE inhibition activity (82 § 0.14%).
DH is a measure of the extent of hydrolytic degradation of a
protein. It is considered the most practical and convenient
means for measuring the hydrolysis process. The DH of
apricot kernel protein Alcalase® hydrolysate increased with
the increase in hydrolysis time (data not shown). It is worth
noting that the highest DH did not correspond to the highest
ACE inhibitory activity. The results indicate that there
exists an optimal hydrolysis point or degree of hydrolysis.
This may be due to the fact that ACE inhibitory peptides
were hydrolyzed to produce inactive small peptides or
amino acids when further increasing hydrolysis time. Other
authors [25] reported that chickpea protein ACE inhibitory
fragments became a target of the enzyme and were also
hydrolyzed with the increase in hydrolysis time.
Molecular weight distribution of AKPH having the highest
ACE inhibition activity
Figure 3 presented the molecular weight distribution of
AKP hydrolyzed for 60 min (i.e., the highest ACE inhibition
activity). The chromatographic data indicated that this frac-
tion was composed of low-molecular weight peptides with
major peaks corresponding to molecules with a less than
1,000 Da molecular weight (Fig. 3). A number of studies
had already shown that the ACE inhibition activity of
150 180 hydrolysates is mainly depending on their molecular weight
distribution [16, 26]. Results of Fig. 3 revealed that the pep-
tide fraction with molecular weight ranging from 200 to
900 Da, representing peptides with two to eight amino acid
residues, was probably associated with higher ACE inhibi-
tion activity. These Wndings are in agreement with observa-
tions from other studies [9] and support the fact that
functional properties of ACE inhibitory peptide are obvi-
ously inXuenced by properties such as molecular mass [27].
EVect of diVerent ultraWltration membranes on IC50
of AKPH
Table 3 presents the in vitro ACE inhibitory activity of the
diVerent studied products. The activity of AKPHIII fraction
Table 3 In vitro ACE inhibitory activity of apricot kernel protein
hydrolysate (AKPH), retentate (AKPHI fraction >5,000 Da), AKPHII
(1,000–5,000 Da) fraction, and permeate (AKPHIII fraction
<1,000 Da) after ultraWltration through a 5,000- and 1,000-Da mem-
brane, respectively
IC50(mg mL¡1)a
AKPH 0.378 § 0.015
AKPHI > 5,000 Da 0.849 § 0.013
AKPHII 1,000–5,000 Da 0.601 § 0.009
AKPHIII < 1,000 Da 0.15 § 0.007
Captopril (as a control) 7.5 £ 10¡6 § 2.1 £ 10¡7
a
IC50 = concentration needed to inhibit 50% ACE activity (expressed
as the mean of three determinations § SD)

123
18 Eur Food Res Technol (2010) 231:13–19

1.4
1 2
Absorbance at 280nm

1.2
1
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120
Fraction number

Fig. 4 Sephadex G15 chromatographic proWle of AKPH fraction

<1,000 Da. Separation was performed with 0.1% TFA in water at a
Xow rate of 0.5 mL min¡1, and 3-mL fractions were collected. The
eZuent was monitored using an ultraviolet detector at 280 nm

(<1 kDa, IC50 = 0.15 mg mL¡1) was 2.5 times higher than
that found in the AKPH and more than Wve times higher
than that measured in the AKPHI fraction (>5 kDa). This
indicated that ACE inhibition was mainly attributable to
peptide components with molecular masses lower than
1,000 Da. This Wnding is in accordance with our result of
molecular weight distribution. Therefore, ultraWltration
through membranes with 1,000 Da of molecular mass
cutoV could be used to obtain a product enriched with ACE
inhibitory peptides.
The most economic way will be to market the peptides
as contained in the crude protein hydrolysate with a speciWc
molecular weight range. Until now, membrane separation
techniques have provided the best technology available for
the enrichment of peptides with a speciWc molecular weight
range [28]. UltraWltration is widely used to enrich bioactive
peptides from various protein hydrolysates such as soy pro-
tein [15], Wsh protein [26], and alfalfa white protein con-
centrate [29]. It is also important to note that membrane
separation, especially ultraWltration,is an industrial-scale
technology suitable for ACE inhibitory peptides enrich-
ment.
Fig. 5 RP-HPLC chromatograms of (a) AKPH fraction <1,000 Da (b)
fraction 1 from Sephadex G15 column chromatography (c) fraction 2
PuriWcation and characterization of ACE inhibitory from Sephadex G15 column chromatography. Elution was achieved
peptides with a linear gradient of acetonitrile (10–25%) + 0.07% TFA and water
(90–75%) + 0.1% TFA at a 1.0 mL min¡1 Xow rate
For the puriWcation of ACE inhibitory peptides, a 60-min
Alcalase® protein hydrolysate was Wrst Wltered through a
1,000-Da membrane with an ultraWltration system. The per- Figure 5a shows the RP-HPLC chromatogram of
meate was then lyophilized and concentrated in distilled AKPHIII fraction (<1,000 Da). It can be seen that the
water. It was loaded onto a Sephadex G15 column to purify hydrolysate was still a complex mixture after Wltration
the hydrolysate. Figure 4 showed the Sephadex G15 gel through a 1,000-Da cutoV membrane. Figure 5b and c pres-
Wltration chromatography proWle of AKPHIII fraction ent the RP-HPLC chromatograms of fractions 1 and 2 from
(<1,000 Da). From this result, AKPHIII fraction was Sephadex G-15 column. Interestingly, the major peaks of
divided into two fractions, and then the two fractions were fractions 1 and 2 were eluted before and after 15 min,
further characterized by RP-HPLC C18 column, respec- respectively (Fig. 5b and c). Fraction 2 with peaks eluting
tively. at longer retention times possessed higher ACE inhibitory

123
Eur Food Res Technol (2010) 231:13–19
activity than fraction 1 (data not shown). Although there is
no clear relationship between the peptide structure and
ACE inhibitory activity, it is also important to point out that
ACE inhibitory peptides are usually constituted by hydro-
phobic amino acids [27, 30], which will increase the reten-
tion time and delay elution. This idea had also previously
been proposed by other research groups [24, 31].
Conclusions
In conclusion, the protease Alcalase® is very well suited for
the generation of apricot kernel protein hydrolysates
enriched with ACE inhibitory peptides. A 60-min Alca-
lase® protein hydrolysate, with the peptides fraction with a
molecular weight mainly ranging from 200 to 900 Da,
showed the highest ACE inhibitory activity (82 § 0.14%).
Moreover, ultraWltration through 1-kDa cutoV membranes
was successfully employed to enrich ACE inhibitory pep-
tides. In addition, puriWcation and characterization of ACE
inhibitory peptides were performed by gel Wltration chro-
matography using Sephadex G15 and C18 RP-HPLC. The
present results have shown that apricot kernel protein is a
good raw material for enzyme-mediated production of ACE
inhibitory peptides. Further isolation and identiWcation of
speciWc peptides with ACE inhibitory activity, elucidation
of the relationship between peptide structure and activity,
and its antihypertensive eVect in vivo await future study.
Acknowledgments This work has received Wnancial support from
project 2009JM 3021 (Shaanxi Province Basic Science Research). The
authors express their appreciation to Professor Dai Jun (Jiangnan Uni-
versity) to determine peptides molecular weight distribution.
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