Seminar on Graduate Institute of Food Science and Biotechnology of National Chung Hsing
University
Title: Purification and characterization of angiotensin I-converting enzyme inhibitory peptides of small red
bean (Phaseolus vulgaris) hydrolysates
Speaker: Pei-Chun Chen (NO. 2)
(陳姵均 7101043024)
Moderator: Nan-Nong Sun (孫楠辳 7101043035)
Advisor: Prof. Chi-Fai Chau
Date: 22nd Nov. 2013
1. Introduction
Hypertension is a popular disease in the world and is considered as the leading risk factor for mortality.
Angiotensin I-converting enzyme (ACE) is an important factor in the regulation of blood pressure. It will
lead to the raise of blood pressure by catalyzing the hydrolysis of angiotensin I to angiotensin II, a potent
vasoconstrictor. Although the synthetic ACE inhibitors (e.g., Captopril, Lisinopril) do benefit the patients,
the significant adverse effects such as cough, taste alterations, skin rashes disturb us. Due to these side
effects, the researchers try to identify and extract ACE inhibitors from natural food sources. Recent
research indicated that various peptides can inhibit ACE. Generally, they were released from different
proteins sources such as casein, fish and cereals.
Dry bean (Phaseolus vulgaris), is a staple food in the human diet. Most dry bean varieties contain 2030% protein, which is twice higher than what is found in cereal grains (7-14%). The small red bean is one
of the varieties of dry beans, which was demonstrated the hydrolysates using alcalase/papain digestion
have higher ACE inhibitory activity. In this study, the authors try to purify the ACE inhibitory peptides
and give the detailed theoretic knowledge for the purification process which can be expanded to other pulse
legumes.
2. Materials and methods
I. Materials:
Small red bean (Phaseolus vulgaris), alcalase (2.4 AU/g), papain, pepsin, trypsin, α-chymotrypsin,
Angiotensin I-converting enzyme (ACE), ACE substrate: Hippuryl-His-Leu (HHL).
Ⅱ. Methods:
i.
Protein extraction
Small red bean protein was extracted with Millipore water and shaken for 1 hr at 55 °C with the pH
adjusted to 9.
ii. Protein hydrolysis
A. Protein hydrolysis used sequential digestion by alcalase and papain. The supernatants will conduct
the in vitro gastrointestinal simulation digestion (GIS).
B. GIS was conducted by sequential digestion by pepsin, trypsin and α-chymotrypsin.
iii. Assay of ACE inhibitory activity by RP-HPLC at OD228:
iv.
Kinetics of inhibition
Inhibition type was determined by plotted Lineweaver-Burk graphs of reciprocal of HA absorbance
versus reciprocal of HHL concentrations.
v. Amino acids sequence determination
Measured by electrospray mass spectrometry (ES MS/MS), and was then analyzed by Xcalibur
software.
3. Result and discussion
I. Enzymatic hydrolysis
i. The most abundant peptides of the hydrolysates were eluted at 26.8min, which corresponded to a
molecular mass (MM) around 0.698 KDa. The result showed that the digestion treatment do help to
break down the large protein.
ii. The IC50 value of the hydrolysates was 67.2 ± 1.8 μg protein/mL. It indicated that the enzymatic
digestion process used in the study can release ACE inhibitory peptides from the small red bean.
Ⅱ. Purification of ACE inhibitory peptides
i. Ultrafiltration used 5 KDa as molecular weight cut off. The permeate fraction with small molecular
weight was determined to have higher ACEI ability. The IC50 value is 50.8 ± 4.4 μg protein/mL, and is
1.3 times lower than the original one.
ii. In the gel filtration, there are 8 fractions were collected. Based on the ACEI activity and molecular
weight, the fraction 5 were collected with IC50 value 35.1 ±1.8μg protein/mL.
iii. The result of RP-HPLC showed that the F5 fraction was consisted of mixture of peptides. Most of
them inhibited more than 50% of ACE activity.
iv. Fraction 28 which eluted at around 30% of acetonitrile proved the most potent ACEI with IC50
value19.3 ±1.4 μg protein/mL.
v. The Lineweaver-Burk plot showed that the Vmax is unchanged by the presence of the competitive
inhibitor demonstrating that the model is competitive inhibition.
vi. The inhibition constant (Ki) value of fraction 28 was 11.6 ± 1.7 μg protein/mL. Lower Ki values
indicate higher affinity.
Ⅲ. Mass spectrometric characterization of selected peptide fraction
i. The presence of octapeptide PVNNPQIH showed the highest frequency. This peptide may be the
predominant fraction of potent ACEI peptide.
ii. The octapeptide PVNNPQIH originated from the major storage protein phaseolin.
iii. The peptide was synthesized and demonstrated an IC50 value 206.7 ±3.9 μM.
4. Conclusions
i. A potent ACEI fraction was collected by three steps purification process which showed competitive
inhibition.
ii. The result showed the small red bean protein could be the good source of ACE inhibitors.
iii. The study provided the detailed method to purify the potent ACEI peptide and may be applied to other
legumes sources.
5. Reference
1. * Rui, Xin, Boye, Joyce I., Simpson, Benjamin K., Prasher Shiv O. (2013). Purification and characterization
of angiotensin I-converting enzyme inhibitory peptides of small red bean (Phaseolus vulgaris) hydrolysates.
Journal of Funtional Foods, 5(3), 1116-1124.