Plant Foods Hum Nutr (2009) 64:8693

DOI 10.1007/s11130-009-0108-1

ORIGINAL PAPER

Sunflower Protein Hydrolysates Reduce Cholesterol

Micellar Solubility
Cristina Megas & Justo Pedroche & Mara del Mar Yust &
Manuel Alaiz & Julio Girn-Calle & Francisco Milln &
Javier Vioque

Published online: 10 February 2009

# Springer Science + Business Media, LLC 2009

Abstract Plant protein hydrolysates are a source of

bioactive peptides. There are peptides that decrease the
micellar cholesterol solubility from bile acids and therefore
may reduce in vivo cholesterol absorption. The presence of
these peptides in sunflower protein hydrolysates has been
studied. Sunflower protein hydrolysates produced with
alcalase plus flavourzyme or with pepsin plus pancreatin
inhibited in some degree the cholesterol incorporation to
micelles. Protein hydrolysates generated after 30 min of
hydrolysis with alcalase, and after 30 min of hydrolysis
with pepsin, were the inhibitoriest of the cholesterol
incorporation to micelles. The average amino acid hydrophobicity of inhibitory peptides in cholesterol micelles was
higher than the observed in the corresponding protein
hydrolysates. This high hydrophobicity probably favours
their inclusion in the lipid micelles. In vivo, this inhibition
may translate in a decrease of cholesterol absorption.
Reported results show that a combination of different characteristics such as peptide size or hydrophobicity may be
responsible of the inhibitory activity of generated peptides.
Keywords Sunflower . Protein hydrolysate .
Bioactive peptides . Cholesterol solubility

Introduction
The development of new foods using low-cost agricultural
by-products is of great nutritional and economical interest.
C. Megas : J. Pedroche : M. del Mar Yust : M. Alaiz :
J. Girn-Calle : F. Milln : J. Vioque (*)
Instituto de la Grasa (C.S.I.C.),
Padre Garca Tejero 4,
41012 Sevilla, Spain
e-mail: jvioque@cica.es

An example of this is the production of protein isolates and

hydrolysates from defatted flours from oilseed crops such
as rapeseed [12] or sunflower [3]. Protein hydrolysates
have many applications in the food industry. Thus, protein
hydrolysates with a low degree of hydrolysis are used to
improve functional properties of foods, and extensive
protein hydrolysates are employed as protein supplements,
or in specialized nutrition such as hypoallergenic foods [4].
In recent years, protein hydrolysates are also considered
a source of bioactive peptides. These are small amino acid
sequences in food proteins that have beneficial biological
activity, after they are released during gastrointestinal
digestion or by previous in vitro protein hydrolysis [5].
Bioactive peptides from plant and animal proteins with
different functions have been obtained, including immune,
opioid, metal chelating activity and inhibition of the
angiotensin converting enzyme (ACE) [6].
Some bioactive peptides have hypocholesterolemic activity.
Cholesterol is a water-insoluble molecule and its intestinal
absorption has a complexity similar to other lipids, including a
previous step of micellar solubilisation [7]. It has been
suggested that hypocholesterolemic peptides inhibited micellar solubility of cholesterol, decreasing in vivo cholesterol
absorption [8]. This class of peptides has been produced after
plant [9-11] and animal [8] protein hydrolysis.
Sunflower protein isolates have been used as substrates
for the production of protein hydrolysates [3], and bioactive
peptides with ACE inhibitory or metal chelating activity,
and have been generated after sunflower protein hydrolysis
with different proteases [1214].
In this work, we have studied the generation of
inhibitory peptides of cholesterol incorporation to micelles,
after the hydrolysis of sunflower proteins with bacterial
proteases (alcalase and flavourzyme) or digestive enzymes
(pepsin and pancreatin).

Plant Foods Hum Nutr (2009) 64:8693

87

Materials and Methods

Degree of Hydrolysis

Materials

The degree of hydrolysis was calculated by determination

of free amino groups by reaction with TNBS [16]. The total
number of amino groups was determined in a sample of
protein isolate hydrolyzed by treatment with 6 N HCl at
120 C for 24 h.

Defatted sunflower meal was supplied by MIGASA (Sevilla,

Spain). Alcalase 2.4 L and flavourzyme 1000 MG were
purchased from Novo-Nordisk (Bagsvaerd, Denmark). Pepsin,
pancreatin, sodium taurocholate, cholesterol, oleic acid and
trinitrobenzenesulfonic acid (TNBS) were from Sigma (Tres
Cantos, Madrid, Spain). All other chemicals were of analytical
grade.

Determination of Inhibition by Sunflower Peptides

of Cholesterol Incorporation to Bile Salts Micelles

Sunflower protein isolates were prepared according to Lqari

et al. [15] with modifications. Briefly, sunflower defatted
meal (20 g) was extracted by stirring for 1 h in 200 mL of
0.2% NaOH pH 12. After centrifugation at 8,000g for
20 min, two additional extractions were carried out with
half the volume of alkaline solution. The pH of the
supernatant was adjusted to the isoelectric point (pH 4.3)
of sunflower proteins, and the precipitate formed was
recovered by centrifugation as described above. The
precipitate was washed with distilled water adjusted to the
isoelectric point and freeze-dried until further use.

The in vitro micellar solubility of cholesterol in the

presence of sunflower protein hydrolysates was measured
according to the method of Nagaoka et al. [8] with some
modifications. Micellar solutions (1 mL) containing 10 mM
sodium taurocholate, 0.4 mM cholesterol, 1 mM oleic acid,
132 mM NaCl, 15 mM sodium phosphate (pH 7.4), and 1
mg of samples were prepared by sonication. These micellar
suspensions were incubated at 37 C for 24 h and then
ultracentrifuged at 38,000 rpm for 60 min at 37 C.
Cholesterol not incorporated to micelles that remain in the
supernatants obtained after ultracentrifugation were
extracted three times with chloroform (3 mL each) which
was later evaporated by flushing with nitrogen for
determination of cholesterol by gas chromatography.

Preparation of Sunflower Protein Hydrolysates

Fractionation of Sunflower Inhibitory Peptides

Sunflower protein hydrolysates were prepared according to

Villanueva et al. [3] with modifications. Briefly, protein
isolates were hydrolyzed using a 1,000 mL hydrolysis
reactor vessel equipped with a stirrer, thermometer, and a
pH electrode. Proteins were digested first for 60 min with
alcalase. Then flavourzyme was added and protein digested
for another 120 min with both enzymes. Hydrolysis parameters were as follows: sunflower protein isolate concentration,
10% (w/v); enzyme/substrate ratio, 0.3 Anson units g1;
pH 8; temperature, 50 C for alcalase and enzyme/substrate
ratio 100 LAPU g1; pH 7; temperature, 50 C for
flavourzyme. Hydrolysis was stopped by heating at 80 C
for 20 min. Hydrolysates were clarified by ultrafiltration
through 0.45 m filters (Millipore, Bedford, MA) to remove
insoluble substrate and lyophilized for storage at 20 C.
Protein isolates were also hydrolyzed by sequential treatment with pepsin and pancreatin. A digestion with pepsin for
180 min was followed by incubation with pancreatin for
another 180 min. Hydrolysis parameters were used as follows:
protein isolate concentration, 10% (w/v); enzyme/substrate
ratio, 1:20 (w/w); pH 2 for pepsin and 7.5 for pancreatin;
temperature, 37 C. Hydrolysis was stopped by heating at 80 C
for 20 min. Hydrolysates were clarified by ultrafiltration
through 0.45 m filters (Millipore, Bedford, MA, USA) to
remove insoluble substrate and lyophylized.

Pellet obtained after ultracentrifugation at 38,000 rpm for

60 min was extracted with a 1/1 (v/v) solution of water
(containing 0.1% trifluoroacetic acid) and chloroform. After
extraction, samples were centrifuged in eppendorf tubes at
12,000 rpm for 10 min, and three different fractions were
generated: an aqueous upper fraction containing the water
soluble peptides, a lower chloroformic phase containing the
lipids from micelles, and an intermediate fraction between
the two above containing non water soluble peptides and
other water insoluble compounds. The upper water fraction
and the intermediate fraction were recovered for the
determination of peptide contents and their amino acid
composition.

Preparation of Sunflower Protein Isolates

Cholesterol Content Determination

Cholesterol content in supernatants obtained after ultracentrifugation was determined by gas chromatography. For
this, a Hewlett-Packard GC 5890 model series II, fitted
with a flame ionization detector and a HP 3390A integrator
(Palo Alto, CA, USA) was used. Hydrogen at 12 psi
column head pressure and 1 mL/min flow rate was
employed as the carrier gas. Nitrogen was used as an
auxiliary gas. Cholesterol was derivatized using a mixture
of pyridine: hexamethyldisilane: trimethylchlorosilane

88

Amino Acid Analysis

Samples were hydrolyzed by incubation in 6 N HCl at
110 C for 24 h in tubes sealed under nitrogen. Amino
acids were determined in the acid hydrolyzate by highperformance liquid chromatography according to the
method of Alaiz et al. [17].

70
60

Degree of hydrolysis (%)

(9:3:1) at room temperature for 15 min. Gas chromatography analyses were performed using a HP-5 25 m0.32
mm0.17 m capillary column. Injector and detector
temperature were maintained at 325 C. The oven temperature was maintained at 255 C.

Plant Foods Hum Nutr (2009) 64:8693

50
40
30
20
10
0
0

Stability to Gastrointestinal Proteases of Inhibitory Peptides

Generated with Alcalase
For simulation of the gastric fluids digestion, sunflower
peptides were incubated for 1 h at 37 C in a 0.32% (w/v)
pepsin solution containing 30 mM NaCl, and 0.7% v/v of
0.2 N HCl, pH 1.2 in a 1/20 (w/w) enzyme/protein
hydrolysate relation.
For simulation of the intestinal fluid digestion, sunflower
peptides were incubated for 3 h at 37 C in a 1% (w/v)
pancreatin solution containing 0.05 M KH2PO4 and 19%
(v/v) 0.2 N NaOH, pH 7.5 in a 1/20 (w/w) enzyme/protein
hydrolysate relation.

Results and Discussion

Production of Protein Hydrolysates
Sunflower protein hydrolysates were obtained by treatment
with the endoprotease alcalase and the endo/exoprotease
flavourzyme. These microbial proteases are used in the
food industry in order to improve the functional and
nutritional properties of protein preparations. Hydrolysis
by alcalase increases very fast in the initial minutes and it
was essentially over after 30 minutes, but hydrolysis by
flavourzyme took longer (Fig. 1). After one hour, alcalase
yielded a protein hydrolysate with a 27.7% degree of
hydrolysis. The final protein hydrolysate obtained after the
digestion of sunflower proteins with alcalase and flavourzyme possessed a 66.6% degree of hydrolysis. This
procedure yields degrees of hydrolysis higher than those
obtained using either alcalase or flavourzyme alone [3].
Pepsin and pancreatin were also used to produce sunflower
protein hydrolysates. Pepsin is the main proteolytic enzyme
generated in the stomach during food digestion. Pancreatin
includes proteases, such as trypsin, chymotrypsin, and
elastase that are released by the pancreas in the small bowel.
Thus, the final sunflower protein hydrolysate obtained after

25

50

75

100

125

150

175

Time (min)

Fig. 1 Time course of the hydrolysis of sunflower protein isolate by

alcalase (added at time 0 min) and flavourzyme (added 60 min later).
Data correspond to the averageSD of three determinations

hydrolysis with pepsin and pancreatin represents a pool of

peptides that may resemble those generated during the
digestion of sunflower proteins in the organism. Figure 2
shows the kinetics of the hydrolysis of sunflower protein
isolate by pepsin and pancreatin. The rate of hydrolysis with
pepsin is very high in the first 10 min, and after that
hydrolysis proceeds slowly. On the other hand, pancreatin
activity is observed even after 3 h of incubation with the
protease. Most likely, the sequential action of the different
proteases included in the pancreatin extract extends the
hydrolysis over a longer time. After 3 h, pepsin yielded a
protein hydrolysate with a 15.5% degree of hydrolysis. The
final protein hydrolysate obtained after digestion of sunflower proteins with pepsin and pancreatin possessed a 37%
degree of hydrolysis.
Inhibition of Cholesterol Incorporation to Micelles
by Sunflower Protein Hydrolysates
Protein hydrolysates produced with alcalase plus flavourzyme
inhibited the incorporation of cholesterol to the micelles
(Fig. 3). Thus, inhibition increases with time and degree of
hydrolysis up to 30 min of hydrolysis with alcalase. Addition
of flavourzyme results in a new decrease of micellar
cholesterol up to 100 min of hydrolysis with this protease.
Protein hydrolysate obtained after 30 min of hydrolysis with
alcalase provided the highest inhibition of cholesterol
incorporation to micelles. This hydrolysate was selected for
further characterization of its inhibitory peptides.
Hydrolysis with pepsin plus pancreatin yielded protein
hydrolysates that inhibited cholesterol incorporation to
micelles, in a higher extent than hydrolysates obtained with
alcalase plus flavourzyme (Fig. 4). Inhibition of cholesterol

Plant Foods Hum Nutr (2009) 64:8693

89

40

solubility using 5 mg/mL of a soy protein hydrolysate. The

hydrolysate with the highest inhibition of cholesterol
incorporation to micelles, obtained after 30 min of
hydrolysis with pepsin, and the final hydrolysate, obtained
after extensive hydrolysis with pepsin plus pancreatin, were
selected for further characterization of their peptides.

Degree of hydrolysis (%)

35
30
25
20
15
10

Stability to Gastrointestinal Proteases of Inhibitory Peptides

Obtained after 30 min of hydrolysis of Sunflower Proteins
with Alcalase

5
0
0

50

100

150
200
Time (min)

250

300

350

Fig. 2 Time course of the hydrolysis of sunflower protein isolate by

pepsin (added at time 0 min) and pancreatin (added 180 min later).
Data correspond to the averageSD of three determinations

incorporation to micelles does not appear to be related to

the degree of hydrolysis. Hence, hydrolysates with the
highest inhibitory activity were obtained after 30 min, 190
min and 360 min of hydrolysis with pepsin plus pancreatin.
These hydrolysates possessed degrees of hydrolysis of
10.1%, 24% and 37%, respectively, and reduced the
cholesterol micellar concentration by 72.3%, 64.2% and
60%, respectively, with respect to control and with 1 mg/
mL peptides concentration.
These results improved those obtained by other authors.
For example, Nagaoka et al. [8] obtained 20% reductions in
micellar cholesterol using 10 mg/mL of a casein tryptic
hydrolysate. Also, a serum bovine -lactoglobulin hydrolysate, in a 10 mg/mL concentration, reduces the cholesterol micellar solubility by 38% [8]. Finally, Nagaoka et al.
[18] obtained a 68% reduction in the cholesterol micellar

Amino Acid Composition of Inhibitory Peptides

The amino acid composition of the original sunflower protein
hydrolysates, the inhibitory peptides contained in micelles
and the water soluble and insoluble peptides extracted from
these micelles were studied (Tables 1, 2, and 3).

100
90

Micellar cholesterol (% of control)

Fig. 3 In vitro inhibition of

cholesterol incorporation to
micelles by sunflower protein
hydrolysates obtained with alcalase plus flavourzime

To study the resistance of these peptides to digestion in the

stomach and small bowel, they were incubated with simulated
gastric (SGF) and intestinal fluids (SIF). As result of the
incubation with SGF, the inhibition of cholesterol incorporation to micelles by these peptides decreased proportionally
with the time of incubation up to 60 min (Fig. 5). However,
when peptides incubated with SGF were further treated with
SIF, a new decrease in the cholesterol micellar solubility was
observed. This decrease in the cholesterol micellar solubility
with the incubation time during intestinal digestion, is also
accompanied by a progressive increase in the degree of
hydrolysis of the sample, suggesting that during the
incubation with SFI new inhibitory peptides are generated
that increase the inhibitory activity of the hydrolysates.

80
70
60
50
40
30
20
10
0

10

15

20

30

40

50

60

70

80

90 100 120 140 160 180

Sunflower protein hydrolysates (min of hydrolysis)

90

100
90
Micellar cholesterol (% of control)

Fig. 4 In vitro inhibition of

cholesterol incorporation to
micelles by sunflower protein
hydrolysates obtained with
pepsin plus pancreatin

Plant Foods Hum Nutr (2009) 64:8693

80
70
60
50
40
30
20
10
0
0

20 30 45 60 90 120 150 180 190 200 210 225 240 270 300 330 360
Sunflower protein hydrolysates (min of hydrolysis)

From the amino acid composition of each peptide

fractions, the hydrophobicity average of these fractions
was calculated using the hydrophobic values for each
amino acid suggested by Tossavainen et al. [19] (Fig. 6).
In all cases, peptides contained in micelles showed higher
average hydrophobicity than the original protein hydrolysates produced with alcalase, pepsin or pancreatin. These
peptides had higher contents of certain hydrophobic amino
acids in relation to the original protein hydrolysate. Thus,
higher amounts of Ala, Tyr, Val, Leu or Lys are observed in
inhibitory peptides. Possibly the higher hydrophobicity
favours the immersion of these peptides in the lipid
micelles. These peptides were further fractionated in water
100

70

% Micellar cholesterol
% Degree of hydrolysis

65

80

60

70

55

60

50

50

45

40

40

30

35

20

30

10

25

20

Degree of hydrolysis (%)

90

Micellar cholesterol (%)

Fig. 5 Inhibitory activity of sunflower peptides obtained after

30 min of hydrolysis with alcalase
after incubation in simulated gastric fluid (SGF) for 60 min and
simulated intestinal fluid (SIF) for
180 min. Numbers in the x-axis
indicate time of incubation in
SGF or SGF plus SIF

soluble and water insoluble peptides (bars 3 and 4,

respectively in Fig. 6). As expected, water soluble peptides
were less hydrophobic than water insoluble ones. Also,
water insoluble peptides showed higher proportion of
hydrophobic amino acids such as Ala, Tyr, Val, Leu or Lys
plus Ile and Phe in relation to water soluble peptides.
The sunflower protein hydrolysate obtained with alcalase,
with the highest inhibitory activity of cholesterol micellar
solubility, also has the greatest hydrophobicity (Table 4).
However, hydrophobicity is not the only factor in determining the inhibitory activity of these peptides. Hence,
pepsin (30 min) and pancreatin (360 min) protein hydrolysates, with the same hydrophobicity average yielded a

SGF 1 h + SIF 180'

SGF 1 h + SIF 120'

SGF 1 h + SIF 90'

SGF 1 h + SIF 60'

SGF 1 h + SIF 30'

SGF 1 h + SIF 15'

SGF 1 h + SIF 0'

SGF 60'

SGF 50'

SGF 40'

SGF 30'

SGF 20'

SGF 10'

SGF 0'

Plant Foods Hum Nutr (2009) 64:8693

91

Table 1 Percent amino acid composition of protein hydrolysate obtained after 30 min of hydrolysis with alcalase, peptides from this hydrolysate
contained in cholesterol micelles, and water soluble and insoluble peptides extracted from the micelles
Amino acids

Aspb
Gluc
Ser
His
Gly
Thr
Arg
Ala
Tyr
Val
Met
Cys
Ile
Leu
Phe
Lys

Hydrophobicity
(kJ/mol)a

Protein hydrolysate
(g/100 g)

Inhibitory peptides
(g/100 g)

Water soluble peptides

(g/100 g)

Water insoluble peptides

(g/100 g)

2.25
2.30
0.17
2.10
0.00
1.85
3.10
3.10
12.0
7.05
5.45
4.20
12.4
10.1
11.1
6.25

11.380.26
24.100.55
5.380.12
2.480.06
6.620.15
4.440.10
9.820.23
5.060.12
2.270.05
4.960.11
0.830.02
0.930.02
4.030.09
7.760.18
6.720.15
3.210.07

12.120.25
16.970.36
4.650.09
2.320.05
6.660.14
5.450.12
6.660.14
9.290.20
2.520.05
6.260.13
0.210.00
0.600.01
4.340.09
9.890.21
4.940.10
7.070.15

12.550.30
31.210.75
4.800.12
1.850.04
7.420.18
5.020.12
6.550.16
7.950.19
2.510.06
4.250.10
0.000.00
0.330.01
3.160.08
7.420.18
4.470.11
0.660.02

11.360.23
14.150.28
5.870.12
2.250.05
6.540.13
6.000.12
6.220.12
9.650.19
4.180.08
6.330.13
0.320.01
0.210.00
4.070.08
10.510.21
5.360.11
6.970.14

Data are the average of two independent determinationsSD

Hydrophobicity values according to Tossavainen et al. [19]
b
Aspartic acid + asparagine
c
Glutamic acid + glutamine
a

quite different inhibition of cholesterol incorporation to

micelles. Other factors, such as the peptide size, may be
critical for this inhibitory activity.
Our findings are consistent with those of other authors.
For example, Zhong et al. [10] fractionated a soy protein

hydrolysate obtained with alcalase, which presented a high

inhibitory activity of cholesterol micellar solubility. They
observed that the inhibition of the cholesterol micellar
solubility increased with increasing hydrophobicity of
collected fractions. Also, Kwon et al. [20] compared the

Table 2 Percent amino acid composition of protein hydrolysate obtained after 30 min of hydrolysis with pepsin, peptides from this hydrolysate
contained in cholesterol micelles, and water soluble and insoluble peptides extracted from the micelles
Amino acids

Aspb
Gluc
Ser
His
Gly
Thr
Arg
Ala
Tyr
Val
Met
Cys
Ile
Leu
Phe
Lys

Hydrophobicitya
(kJ/mol)

Protein hydrolysate
(g/100 g)

Inhibitory peptides
(g/100 g)

Water soluble peptides

(g /100 g)

Water insoluble peptides

(g/100 g)

2.25
2.30
0.17
2.10
0.00
1.85
3.10
3.10
12.0
7.05
5.45
4.20
12.4
10.1
11.1
6.25

11.970.36
27.540.83
4.670.14
1.560.05
6.130.18
4.090.12
10.610.32
4.960.15
1.650.05
5.250.16
1.460.05
0.000.00
4.480.13
7.780.23
5.450.16
2.430.07

11.070.20
15.180.27
5.900.11
2.210.04
7.690.14
6.750.12
6.750.12
11.590.21
2.530.05
5.380.10
0.950.02
0.320.01
4.430.08
8.750.16
4.430.08
6.120.11

8.910.20
16.890.37
6.940.15
1.970.04
8.180.18
6.940.15
5.690.13
11.810.26
3.830.08
5.590.12
0.410.01
0.310.01
4.040.09
8.390.19
4.460.10
5.590.12

8.470.21
13.020.33
7.440.19
2.790.07
7.640.19
5.990.15
8.160.20
8.880.22
4.550.11
6.300.16
0.000.00
0.000.00
4.650.12
10.020.25
5.790.15
6.300.16

Data are the average of two independent determinationsSD

Hydrophobicity values according to Tossavainen et al. [19]
b
Aspartic acid + asparagine
c
Glutamic acid + glutamine
a

92

Plant Foods Hum Nutr (2009) 64:8693

Table 3 Percent amino acid composition of protein hydrolysate obtained after 180 min of hydrolysis with pepsin and 180 min with pancreatin,
peptides from this hydrolysate contained in cholesterol micelles, and water soluble and insoluble peptides from the micelles
Amino acids

Hydrophobicitya
(kJ/mol)

Protein hydrolysate
(g/100 g)

Inhibitory peptides
(g/100 g)

Water soluble peptides

(g/100 g)

Water insoluble peptides

(g/100 g)

2.25
2.30
0.17
2.10
0.00
1.85
3.10
3.10
12.0
7.05
5.45
4.20
12.4
10.1
11.1
6.25

11.970.32
27.540.74
4.670.13
1.560.04
6.130.17
4.090.11
10.610.29
4.960.13
1.650.05
5.250.14
1.460.04
0.000.00
4.480.12
7.780.21
5.450.15
2.430.07

10.780.23
12.120.26
7.090.15
2.260.05
7.390.16
5.960.13
8.520.18
8.420.18
2.870.06
6.160.13
0.820.02
0.310.01
4.720.10
9.960.21
4.920.10
7.700.16

12.230.32
23.220.60
10.670.28
1.880.05
9.620.25
4.920.13
6.280.16
6.060.16
1.780.05
4.710.12
0.210.01
0.940.02
3.240.08
5.960.16
2.720.07
5.540.14

10.970.26
14.890.36
6.940.17
2.350.06
6.720.16
6.270.15
5.380.13
10.080.24
4.480.11
6.160.15
0.110.00
0.110.00
4.260.10
9.740.23
4.930.12
6.940.17

Aspb
Gluc
Ser
His
Gly
Thr
Arg
Ala
Tyr
Val
Met
Cys
Ile
Leu
Phe
Lys

Data are the average of two independent determinationsSD

Hydrophobicity values according to Tossavainen et al. [19]
b
Aspartic acid + asparagine
c
Glutamic acid + glutamine
a

hypocholesterolemic effect of two synthesized peptides

(SPYPR and LPYRR), and concluded that the minor
hypocholesterolemic effect of SPYPR peptide was due to
its lower hydrophobicity. In contrast, Sugano et al. [21]
reported that the inhibitory activity of hypocholesterolemic
peptides did not depend on their hydrophobicity, but on
structural features as its amino acidic sequence.
In conclusion, we show in this work that sunflower
proteins hydrolyzed either with microbial or digestive
proteases represent a source of peptides that inhibits
cholesterol incorporation to micelles. In vivo, this inhibition

may result in a decrease of the cholesterol absorption as has

been reported for soy protein derived peptides [22].
Reported results show that a combination of different
characteristics such as peptide size or hydrophobicity may
be responsible of the inhibitory activity of the generated
peptides.
Table 4 Characteristics of sunflower protein hydrolysates obtained
with alcalase, pepsin and pancreatin and of the corresponding
inhibitory peptides contained in lipid micelles
Protein hydrolysates

Inhibitory

Hydrophobicity average (kJ/mol)

peptides

5
4,8

Degree of

4,6

Hydrophobicityb Micelar

Hydrophobicity

hydrolysisa

cholesterolc of water soluble

(%)

(%)

peptides/water
insoluble

4,4

peptidesd

4,2
4
3,8
3,6

Alcalase (30 min)

32.8

4.35

24.2

Pepsin (30 min)

10.1

4.25

27.7

3.88/4.81
4.4/4.81

Pancreatin

37.0

4.25

40.3

3.59/4.72

(360 min)

3,4
a

3,2
3

Alcalase
hydrolysate

Pepsin
hydrolysate

3
Pancreatin
hydrolysate

Fig. 6 Average hydrophobicity of selected protein hydrolysates

obtained with alcalase, pepsin or pancreatin (1), corresponding
peptides in cholesterol micelles (2), water soluble peptides extracted
from micelles (3), and water insoluble peptides from micelles (4)

Degree of hydrolysis of the sunflower protein hydrolysates

calculated according to Adler-Nissen [16].
b
Hydrophobicity average of protein hydrolysates calculated according
to the values provided by Tossavainen et al. [19]
c
Percent cholesterol in bile salts micelles with respect to the control in
the presence of inhibitory peptides.
d
Hydrophobicity average of water soluble/water insoluble peptides
contained in lipid micelles peptides, calculated according to the values
provided by Tossavainen et al. [19]

Plant Foods Hum Nutr (2009) 64:8693

Acknowledgements This work was supported by research grants
AGL 2004-03930 (F.M.) and AGL 2005-01120 (J.G.-C.) from the
Spanish Ministry of Education and Science, partially supported by
FEDER funds from EU.

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