| Research Article |
Open Access |
|
| Electrophoretic Profiles and Angiotensin I-Converting Enzyme Inhibitory
Activities of Nine Varieties of Phaseolus Vulgaris Protein Hydrolysates |
| Xin Rui1,2, Joyce I. Boye1*, Chockry Barbana1, Benjamin K. Simpson3 and Shiv O. Prasher2 |
| 1Food Research and Development Centre, Agriculture and Agri-Food Canada, 3600 Casavant Blvd. W., St-Hyacinthe, QC, J2S 8E3, Canada |
| 2Department of Bioresource Engineering, Macdonald Campus, McGill University, 21,111 Lakeshore Rd., Ste-Anne-de-Bellevue, QC, H9X 3V9, Canada |
| 3Department of Food Science and Agricultural Chemistry, Macdonald Campus, McGill University, 21,111 Lakeshore Rd., Ste-Anne-de-Bellevue, QC, H9X 3V9, Canada |
| *Corresponding author: |
Joyce Irene Boye
Food Research and Development
Centre
Agriculture and Agri-Food Canada
3600 Casavant Blvd. W., St-Hyacinthe
QC, J2S 8E3, Canada
Tel: +1 450 768 3232
Fax: +1 450 773 8461
E-mail:
Joyce.Boye@agr.gc.ca |
|
| |
| Received July 05, 2011; Accepted August 10, 2012; Published August 13, 2012 |
| |
| Citation: Rui X, Boye JI, Barbana C, Simpson BK, Prasher SO (2012)
Electrophoretic Profiles and Angiotensin I-Converting Enzyme Inhibitory Activities
of Nine Varieties of Phaseolus Vulgaris Protein Hydrolysates. J Nutr Food Sci
2:156. doi:10.4172/2155-9600.1000156 |
| |
| Copyright: © 2012 Rui X, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited. |
| |
| Abstract |
| |
| Nine dry bean (Phaseolus vulgaris) varieties largely grown in Canada were subjected to digestion using
trypsin and in vitro gastrointestinal simulation (GIS) followed by a study of their in vitro ACE inhibitor properties and
digestibility. GIS hydrolysates of all varieties presented significantly higher ACE inhibitory activities and degree of
hydrolysis (DH) compared to those of trypsin hydrolysates (P < 0.05). Cranberry and light red kidney bean protein
isolates contained ‘T’ type phaseolin and had higher DH values during both digestions, with average ACE inhibitory
activities of 281.7–281.8 μg/mL and 141.6–185.1 μg/mL, respectively, for tryptic and GIS hydrolysates. The other
seven bean varieties contained ‘S’ type phaseolin, and of these small red bean showed the lowest ACE inhibitory
activities for both trypsin (IC50 of 170 μg/mL) and GIS (IC50 of 118 μg/mL) digestion, followed by navy bean, with IC50
of 200 μg/mL (trypsin digestion) and 137 μg/mL (GIS digestion). The results demonstrated that both methods of
digestions yielded bioactive peptides, however, differing peptide profiles of the bean protein hydrolysates affected
their in vitro ACE inhibitory property. |
| |
| Keywords |
| |
| Bean protein hydrolysates; Angiotensin I-converting
enzyme; Digestibility; in vitro gastrointestinal digestion simulation;
Trypsin |
| |
| Introduction |
| |
| Peptides with hypotensive properties have received increasing
attention in recent times. Hypertension is ranked as one of the world’s
most common high-risk diseases, affecting about 22% of the world’s
population [1]. Angiotensin I-converting enzyme (ACE, EC 3.4.15.1)
is the key enzyme responsible for elevation of blood pressure as it is
capable of converting the decapeptide, angiotensin I, to the octapeptide,
angiotensin II, a potent vasoconstrictor, and also degrading bradykinin,
a vasodilator, into inactive peptides [2]. Adverse effects resulting from
the use of synthetic ACE inhibitors has enhanced research aimed
at investigating and identifying natural sources of ACE inhibitory
peptides, which may be freer of side effects. |
| |
| Pulses, including pea, lentil, chickpea, and beans contain high
levels of protein, fiber, as well as valuable minerals and vitamins which
enhances their health-benefitting attributes. ACE inhibitory studies
have been conducted on a few pulses, including chickpea [3-5], pea
[5-7], and lentil [8]. However, very few studies have investigated the
antihypertensive properties of beans [9,10]. Canada is one of the most
important global producer and exporter of dry beans and several dry
bean varieties are grown in the country. Unlike other pulses, there is a
dearth of fundamental studies on the ACE inhibitory activities of beans
and more importantly how this activity is affected by varieties. Data on
bean bioactive properties could enable dry bean proteins to be explored
as value added components in functional foods which will provide
benefit to consumers, growers as well as producers. |
| |
| An essential step for the generation of bioactive peptides from
foods is enzymatic hydrolysis. Bean proteins have lower digestibility
compared to animal proteins which can limit their nutritional value
[11], and further affect the potential of generating ACE inhibitory
peptides during hydrolysis. In the current study, the digestibility of
proteins from nine varieties of dry beans (Phaseolus vulgaris) that are
largely cultivated in Canada, namely navy, pink, pinto, cranberry, black,
great northern, light red kidney, dark red kidney, and small red, were studied using trypsin and in vitro gastrointestinal digestion simulation
(GIS). Subsequently, the in vitro ACE inhibitory properties of the
protein digests were analyzed and compared to determine the bean
variety with highest potential for ACE inhibition under the conditions
studied. |
| |
| Materials and Methods |
| |
| Materials |
| |
| Protein isolates of nine varieties of beans (Phaseolus vulgaris),
namely navy, pink, pinto, cranberry, black, great northern, dark
red kidney, light red kidney, and small red beans, were prepared as
described in Rui et al. [12]. Low molecular weight calibration kits
were from Amersham Pharmacia Biotech (Uppsala, Sweden). Precast
10‑20% gradient tris/tricine gels, precast 16.5% tris/tricine gels, tricine
sample buffer, and coomassie brilliant blue G‑250 were from Bio-Rad
Laboratories (Hercules, CA). α-Amylase (A 6380), pepsin (P 6887),
trypsin (T 0303), α-chymotrypsin (C 4129), ACE regent (A 6778),
Hippuryl-His-Leu (HHL) (H 1635) and 2,4,6-trinitrobenzenesulphonic
acid (TNBS) (P 2297) were purchased from Sigma-Aldrich Co.
(Oakville, ON). All other regents used were of analytical grade. |
| |
| Protein analysis |
| |
| Protein/peptide contents in protein hydrolysates were determined according to previous studies [5,13] with the Dumas combustion
method using a LECO FP-428 apparatus (LECO Corp., St. Joseph, MI,
USA) [14]. A factor of 6.25 was used for the conversion of nitrogen to
protein based on the average nitrogen content (16%) of amino acids in
protein mixtures. |
| |
| in vitro protein digestibility: in vitro protein digestibility of bean
protein isolates were determined according to a previously published
method based on measuring pH drop after 10 min of in vitro digestion
[15]. |
| |
| Protein hydrolysis |
| |
| Protein hydrolyses using trypsin and in vitro gastrointestinal
simulation (GIS) digestions were carried out separately in duplicate.
Digestions were conducted using a pH-stat apparatus (TIM 865,
Radiometer Analytical SAS, Villeurbanne, France) with substrate
(concentration of 2.5% w/v, based on protein content) dispersed in
phosphate buffer (0.01 M, pH 8.0) (Trypsin hydrolysis) or glycine buffer
(0.01 M, pH 7.0) (GIS hydrolysis). Trypsin digestions were performed
with an enzyme to substrate ratio (E/S) of 1:25 (w/w, based on protein
content) for 2 h at 37°C. GIS digestions were started by pre-treatment
of the bean protein isolates with α-amylase solution (1 mg/mL, 0.01
M glycine buffer, pH 7.0) at a ratio at 1:12.5 (v/w) at 37°C for 3 min
and then followed by sequential digestions of pepsin, trypsin and
α-chymotrypsin with E/S: 1/250 (w/w, based on protein content) as
described previously in Barbana and Boye [5]. For both trypsin and GIS
digestion, enzyme inactivation after sampling was achieved by heating
in boiling water for 10 min followed by centrifugation at 12,000 g, 4°C
for 20 min. The supernatants were collected and freeze dried for further
electrophoresis and ACE inhibition studies. To follow the time course
of hydrolysis, aliquots of the hydrolysates were withdrawn at different
time intervals during digestion for electrophoretic analysis. The samples
were diluted 10 fold with Laemmli buffer, heated in boiling water for 10
min and stored at -20°C until analysis. |
| |
| Degree of Hydrolysis |
| |
| Degree of hydrolysis (DH, %) was determined based on the
reaction of free amino groups with TNBS [16]. Total number of amino
acid groups was determined by hydrolyzing the samples in 6 M HCl
at 110°C for 24 h. A series concentration of L-Leucine was used to
generate the standard curve. |
| |
| in vitro angiotensin I-converting enzyme inhibitory activity
determination |
| |
| ACE inhibitory activities were determined by using the HPLC
method described previously in Barbana and Boye [5]. A 4.60 x 250
mm Aqua C18 reverse-phase HPLC column (5 μm particle size, 125
Å, Phenomenex) was used. Hippuryl-His-Leu (HHL) was used as
substrate. IC50 values (concentration inhibiting half maximal ACE
activity) were determined by graphed ACE inhibition percentages
versus semi-logarithmic values of sample concentrations. |
| |
| Electrophoresis |
| |
| SDS-PAGE analyses of the bean trypsin hydrolysates were carried
out using a Bio-Rad Criterion Cell (Bio-Rad Laboratories, Inc.,
Mississauga, ON) with 16.5% tris-tricine gels. GIS hydrolysates were
analyzed using 10‑20% gradient tris-HCl gels. For studies under
denaturing conditions, 5% (v/v) β‑mercaptoethanol (β‑Me) was added
to the electrophoresis reagents during sample preparation prior to
sample loading. Electrophoreses were conducted under constant voltage
of 100 V, using Bio-Rad polypeptide standards (1.423–26.625 kDa) and
low-molecular mass standard markers (14.4–97 kDa) calibration kits
from Amersham Pharmacia Biotech for 16.5% tris-tricine gels and
10‑20% gradient tris-HCl gels, respectively. Gels were scanned with a
Bio-Rad GS-690 calibrated imaging densitometer and analyzed using
a Multi-Analyst/PC Analysis software (Bio-Rad Laboratories, Inc.,
Mississauga, ON). |
| |
| Statistical Analysis |
| |
| Analysis of variance (ANOVA) and Duncan’s multiple comparison
tests were used to determine the significant differences between means
(P < 0.05) using SAS Server Interface (version 2.0.3, SAS Institute, Cary,
NC). |
| |
| Results |
| |
| in vitro protein digestibility of the protein isolates extracted from
the nine bean varieties ranged from 83.67% (black bean) to 88.19%
(great northern bean). The highest values were found for navy bean
and great northern bean, at 88.01% and 88.19%, respectively (P < 0.05)
(Table 1). Trypsin digestion of the bean protein isolates gave low degree
of hydrolysis (DH) values, ranging from 1.72% (dark red kidney bean)
to 4.59% (cranberry bean), whereas higher DH values were obtained
after in vitro GIS digestion, ranging from 7.22% (dark red kidney
bean) to 16.12% (pinto bean) (Table 1). Among the nine varieties,
hydrolysates of cranberry, light red kidney and pinto bean for both
methods of digestion exhibited significantly higher DH values than the
other six varieties (P < 0.05). |
| |
|
Table 1: In vitro digestibility (%) of bean protein isolates and degree of hydrolysis (DH, %) of bean proteins hydrolysed using trypsin and in vitro gastrointestinal simulation
(GIS) digestion. |
|
| |
| Under both reducing and non-reducing conditions, SDS-PAGE
profiles of the trypsin hydrolysates of all investigated varieties showed
predominant bands with estimated molecular mass (MM) of 24 kDa
(Figure 1a and b). The intensity of the 24 kDa band was much less for
the hydrolysates of cranberry and light red kidney, but there were more
intense bands at lower MM between 3–6 kDa, and also additional bands
around 16 kDa (lane 4 and 7, Figure 1a and b). The hydrolysate of black
bean had a large amount of a high MM fraction which remained at the
top of the gel (lane 5, Figure 1a and b); this band was absent for navy
and great northern bean hydrolysates (lane 1 and 6, Figure 1a and b). |
| |
|
Figure 1: SDS-PAGE of bean protein trypsin hydrolysates (a, b) and in vitro gastrointestinal simulation (GIS) hydrolysates (c, d): (a, c) non-reducing conditions, (b, d)
reducing conditions (5% β‑ME), Lane 1-9 are 1- navy; 2- pink; 3- pinto; 4- cranberry; 5- black; 6- great northern; 7- light red kidney; 8- dark red kidney; 9- small red. |
|
| |
| Unlike the dry bean trypsin digests, the majority of the bands
observed in the SDS-PAGE after in vitro GIS digestions had a MM
around 47 kDa (Figure 1c and d), and no protein/protein aggregates
were observed at the top of the gel. Additionally, there was not much of
a difference between the samples treated with and without β‑Me. Again, cranberry and light red kidney bean hydrolysates showed different
profiles compared to the seven other varieties (lane 4 and 7, Figure 1c
and d), with the presence of three subunits having MMs of 47, 50, and
53 kDa, whereas the other seven varieties presented only two subunits
profiles with MMs of 46 and 50 kDa. Cranberry and light red kidney
bean hydrolysates also contained smaller proteins having MMs less
than 14 kDa compared to other varieties, and there were fewer bands
between the 24 to 38 kDa range. |
|
| |
| All investigated bean varieties showed ACE inhibitory activities
after trypsin and in vitro GIS digestions. The latter method of hydrolysis
yielded significantly lower IC50 values compared to the former (P < 0.05)
(Figure 2). Lower IC50 values represent higher ACE inhibitory property.
Thus, small red bean hydrolysates from both trypsin and GIS digestions,
had significantly higher (P < 0.05) ACE inhibitory activities compared
to other varieties undergoing similar digestion, with IC50 values of 170
μg/mL and 118 μg/mL, respectively. Navy bean hydrolysates had the
next highest ACE inhibitory activity with IC50 values of 200 μg/mL
(trypsin digestion) and 137 μg/mL (GIS digestion). Varieties presenting
the least ACE inhibitory activities for both digestions were black bean
trypsin hydrolysates (IC50 of 406 μg/mL) and pinto and dark red kidney
GIS hydrolysates (IC50 of 198 μg/mL and 199 μg/mL, respectively). |
| |
|
Figure 2: IC50 values for ACE inhibition properties of bean protein
hydrolysates: 1- navy; 2- pink; 3- pinto; 4- cranberry; 5- black; 6- great
northern; 7- light red kidney; 8- dark red kidney; 9- small red. |
|
| |
| To further understand changes occurring during the hydrolysis
treatment, samples from the navy, black, and small red bean digests were
collected at different time intervals during the digestion and subjected
to electrophoresis (Figure 3a, b, c, d, e and f). The three varieties were
chosen based on the ACE inhibitory results, which showed navy and
small red bean hydrolysates to have high ACE inhibitory activity, and marked improvement in ACE inhibitory activity of black bean
hydrolysates after GIS treatment compared to the trypsin digestion. |
| |
|
Figure 3: SDS-PAGE of bean protein hydrolysates after different time intervals of trypsin hydrolysis (a-navy, b-black, and c-small red); and in vitro gastrointestinal
simulation (GIS) digestion hydrolysates (e-navy, f-black, and g-small red) under reducing conditions (5% β‑ME): lane 1: 0 min; lane 2: 3 min after α-amylase digestion;
lanes 3 to 8: 0 min, 5 min, 10 min, 30 min, 60 min, 120 min of peptic digestion, respectively; lanes 9 to 14: 0 min, 5 min, 10 min, 30 min, 60 min, 150 min of trypsin/α-
chymotrypsin digestion, respectively. |
|
| |
| Minor differences were observed in the electrophoretic profiles
with black bean protein isolates having intense bands in the high MM
range and missing bands in the 60 to 97 kDa MM range (lane 0 min,
Figure 3b). After addition of trypsin, proteins having MM higher
than 30 kDa from all the investigated samples degraded within 5 min
(Figure 3a, b and c) leaving only one persistent faint band with MM of
45 kDa. Two new bands, with MM of around 24 kDa (major) and 28
kDa (minor) appeared which did not degrade any further even after 120
min of hydrolysis (Figure 3a, b and c). |
| |
| The electrophoretic profiles of the samples subjected to GIS
treatment were very similar (Figure 3d, e and f). As expected, pretreatment
with α-amylase did not change the SDS-PAGE profiles (lane
1–2, Figure 3d, e and f). Addition of pepsin had little impact on the
47 kDa protein (lane 3–8, Figure 3d, e and f). However, it led to the
degradation of the proteins with MMs of 20 kDa, 33 kDa, and higher
(>50 kDa). New bands with estimated MM of 22 kDa were generated
for navy and small red bean hydrolysates (lane 3–8, Figure 3d and f).
Subsequent treatments with trypsin and α-chymotrypsin increased the
intensity of the 24 kDa and 28 kDa proteins for all varieties (lane 9–14,
Figure 3d, e and f), whereas the newly formed proteins with MM around
22 kDa generated after the addition of pepsin were gradually degraded
to smaller peptides with the addition of trypsin and α-chymotrypsin
(lane 9–14, Figure 3d and f). |
| |
| Discussion |
| |
| Dry bean proteins are known to be resistant to proteolysis [17].
Various workers have indicated that the predominant protein in dry
beans, i.e. phaseolin, which has a MM of around 47 kDa, has a compact
structure and high β-sheet conformation which makes it resistant to
peptic digestion and partially resistant to trypsin and α-chymotrypsin
digestion [12,18,19]. This is consistent with our findings. The degree of
hydrolysis (DH) value, which represents the extent of hydrolysis, was
very low for all investigated varieties using both methods of digestion.
Previous researchers [5,20] have reported almost three times higher
DH values i.e. 27.08% – 40.78% for other pulses such as chickpea, pea
and lentil subjected to similar GIS digestion. The results, therefore,
suggest that the bean samples analyzed in this study are more resistant
to digestion than some of these other pulses. The limited degree of
hydrolysis did not, however, prevent the enzymatic release of ACE inhibitory peptides from the intact proteins. All investigated dry beans
from both digestion treatments demonstrated comparable in vitro
ACE inhibitory activities to some pulses hydrolysates, such as chickpea
protein GIS digests (IC50:140 – 229 μg/mL) [5], and pea protein GIS
digests (IC50: 70 – 159 μg/mL) [5,7]. The ACE inhibitory activities were,
on the other hand, less than those reported for lentil protein trypsin
digests (IC50: 111 μg/mL) and GIS digests (IC50: 53 – 90 μg/mL) [8,20]. |
| |
| Different enzymatic treatments had varying impacts on the
hydrolysate profiles and the ACE inhibitory properties of the dry beans.
The SDS-PAGE patterns indicated trypsinolysis of phaseolin occurred
very quickly and produced peptides with MM half of the original
sizes. This phenomenon, which is in agreement with other previous
studies [21,22], may be explained by the presence of a hydrophilic
region located near the center of phaseolin which is easily accessible for
proteases to attack [21]. Interestingly, phaseolin was hardly degraded
during GIS digestion probably due to the lower enzyme to substrate
(E/S) ratios used in the GIS digestion, i.e. 1:250 compared to 1:25 used
for trypsinolysis. Previous studies demonstrated minor degradation of
phaseolin during trypsinolysis using an E/S ratio of 1:100; at a higher
E/S ratio of 1:10 phaseolin was halved in 3 min [21,23]. Compared
to trypsin digestion, the significantly higher DH values (P < 0.05)
of the GIS digestion suggests that a larger number of small peptides
were released during the GIS digestion. This was reflected in the
improvement of the ACE inhibitory activities for all investigated dry
bean samples after GIS treatment. |
| |
| Of all the dry bean varieties, distinctive peptide profiles were
observed for the cranberry and light red kidney bean hydrolysates. The
SDS-PAGE profiles suggested that these two bean varieties contained ‘T’
type phaseolin, as categorized by Brown et al. [24], who demonstrated
three phaseolin types, namely, ‘S’, ‘T’, and ‘C’ after the cultivars Sanilac,
Tendergreen, and Contender, respectively. The other seven investigated
bean varieties showed ‘S’ type phaseolin. It appears, therefore, that
dry bean proteins with ‘T’ type phaseolin had higher proteolytic
susceptibility to both digestion treatments, based on the high DH
values obtained for cranberry and light red kidney bean hydrolysates
as well as the intensive bands observed on the SDS-PAGE profiles at
the lower MM range. Digestibility differences between various types of
phaseolin were reported recently [25]. The study showed that ‘T’ type
phaseolin had higher DH values than ‘S’ type phaseolin. Interestingly,
the cranberry and light red kidney bean hydrolysates obtained from
both digestion treatments did not yield significantly higher ACE
inhibitory activities compared to the other seven varieties, even though
they seemed to contain more small peptides. |
| |
| Black bean was particularly different among the samples. DH value
of black bean hydrolysates from GIS digestion was 5.4 times higher
than that of the trypsin digestion, which was the highest among all
the samples. Moreover, the differences between IC50 values from the
two digestions were also the highest for black bean, i.e. 2.7 times. This
observation might be linked to the more intensive degradation of large
protein aggregate in black bean after GIS digestion and the consequent
liberation of larger quantities of small peptides as shown on the SDSPAGE
profiles. |
| |
| A comparison between the digestibility and ACE inhibitory activity
values of the dry bean varieties showed no obvious relationship. As an
example, small red bean, which had the highest ACE inhibitory activities,
had average DH values and similar SDS-PAGE profiles compared to
the other varieties for both digestion treatments. Peptides must meet
several criteria (e.g., shape, molecular mass, hydrophobicity, charge and
electronic properties) in order to yield ACE inhibitory properties [26]. They are expected to be short, i.e. 2 – 12 amino acids [26] and are more
likely to have hydrophobic amino acids as C-terminal residue, such as
tryptophan, tyrosine, phenylalanine and proline [27]. Thus, the ACE
inhibitory activities of the dry bean hydrolysates analysed in this study
may have been influenced not only by the extent of hydrolysis but also
other factors, such as parent protein composition, structure, sequence
and enzymatic digestion mechanisms [5] which opens up avenues for
further studies. |
| |
| In conclusion, the current study investigated for the first time the in vitro ACE inhibitory properties of protein hydrolysates obtained from
nine largely grown Canadian Phaseolus vulgaris bean varieties. We
demonstrated that: a) all investigated bean varieties had ACE inhibitory
activities when subjected to both trypsin and in vitro GIS digestion
treatments; b) all investigated varieties showed significantly higher
ACE inhibitory activities after GIS digestion than after trypsinolysis.
Additionally, the interesting findings for cranberry bean and light red
kidney bean hydrolysates opens up possibilities for future studies about
the relationships between phaseolin types and their bio-functional
properties. More specifically, navy bean and small red bean which
exerted the highest ACE inhibitory activity would be ideal targets
for future studies related to ACE inhibitory properties. Overall, this
manuscript provided novel fundamental knowledge regarding the
ACE inhibitory activity of dry bean and could prove useful for future
applications (e.g., incorporation of dry bean protein hydrolysates from
specific bean varieties in functional foods targeting hypertension). |
| |
| Acknowledgements |
| |
| This project was funded by Agriculture and Agri-Food Canada’s Agricultural
Bio-product Innovation Program. A scholarship from the China Scholarship Council
is also gratefully acknowledged. |
| |
|
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