J Am Oil Chem Soc
DOI 10.1007/s11746-015-2655-y
ORIGINAL PAPER
Amino Acid Profiles of 44 Soybean Lines and ACE-I Inhibitory
Activities of Peptide Fractions from Selected Lines
Srinivas Rayaprolu1 · Navam Hettiarachchy1 · Ronny Horax1 ·
Eswaranandam Satchithanandam2 · Pengyin Chen3 · Andy Mauromoustakos4
Received: 11 August 2014 / Revised: 15 March 2015 / Accepted: 11 April 2015
© AOCS 2015
Abstract Soybeans are cultivated in the United States
chiefly for cooking oil, while the residue after oil extraction
(soybean meal) is mostly used in animal feed formulations.
High protein content in the defatted soybean meals led to
the extraction of pure protein and its application in food
products. We selected 44 soybean lines to determine their
moisture and protein contents, and their amino acid composition was investigated. Soybean lines with high protein
content, one high yielding (R95-1705), and two high oleic
acid (N98-4445A, S03-543CR), were selected for protein
isolate preparation, hydrolysis using alcalase and gastrointestinal (GI) resistance. Furthermore, the GI resistant
hydrolysates were fractionated and tested for angiotensinI-converting enzyme (ACE-I) inhibition activity. The amino
acid analysis showed high methionine in the high protein
and fatty acid lines (R05-4494 and R05-5491), and high
cysteine content in one of the high oleic acid soybean line
CRR05-188 in comparison to the check lines (UA-4805
and 5601-T). The protein isolate with the highest purity
(90–93 %) was derived from the selected lines N98-4445A
and S03-543CR, and hydrolyzed using alcalase enzyme.
The protein hydrolysates (500 µg/mL) showed inhibition
of the ACE-I by 49 %. The results from this study will
* Navam Hettiarachchy
nhettiar@uark.edu
1
Department of Food Science, University of Arkansas,
2650 N Young ave., Fayetteville, AR 72704, USA
2
Department of Chemical Engineering,
University of Arkansas, Fayetteville, AR 72701, USA
3
Department of Crop Soil and Environment,
University of Arkansas, Fayetteville, AR 72701, USA
4
Department of Agricultural Statistics, University of Arkansas,
Fayetteville, AR 72701, USA
promote the use of high oleic acid soybeans as a source of
protein and peptides with functional activities.
Keywords High oleic acid soybean lines · Seed protein ·
Amino acid analysis · Protein hydrolysates · ACE-I
inhibition
Introduction
Soybean is an excellent source of protein and oil and is the
second major crop grown in the United States. The USA is
the largest producer and exporter of soy in the world and
the forecast for 2014 production is approximately 3.3 billion
bushels (~27 kg/bushel) according to the Crop Production
Report by the United States Department of Agriculture [1].
The American Soybean Association and the regional soybean boards have a major role in producing better quality
soybeans using genetic engineering and plant breeding techniques by improving various attributes including enhanced
yields, pest and disease resistance, lipid quality and quantity, and protein content and quality [2, 3]. Soybeans are primarily grown for the edible oil which is separated by various extraction methods, resulting in a leftover residue called
soybean meal. Soybean meal is a major ingredient in animal
feed formulations as a source of complete protein. It is also
a chief source of high-quality plant-based protein in human
diet. Hydrolysates prepared from the soybean proteins are
in great demand as ingredients for food applications as well
as in protein supplements that provide nutritional and health
benefits [4, 5]. Improvements in soybean processing and
functional characteristics have diversified the ever-increasing demand for soy protein ingredients [6, 7].
New soybean lines are produced with higher yields,
higher protein content, and recently with higher amounts
13
J Am Oil Chem Soc
(up to 80 %) of oleic acid in their lipid composition [8].
A higher percentage of monounsaturated fatty acids such
as oleic acid is preferred over polyunsaturated fatty acids
which have lower oxidative stability [9, 10]. Variations in
the plant genes are the cause of differences, not only in
the oil content but also in the concentration of crude protein, possibly even in the amino acid composition [11–13].
Researchers have found an increase in lysine in the hybridized high oleic acid soybean seeds compared to the parent
lines [14, 15]. The amino acid composition of the seed protein depends on the storage protein content, nitrogen supply during the growth phase, and asparagine levels in the
embryonic stage of the plant [16]. There has not been much
research in evaluating the essential amino acid content,
specifically sulfur-containing amino acids in the soy protein from high oleic acid soybean seeds.
Oil-extracted soybean meals contain approximately 1 %
residual oil and have 48 % crude protein with all the essential amino acids required for human health except for the
sulfur amino acids methionine and cysteine [17]. Proteins
have proved to be excellent sources for bioactive peptides,
especially in reducing hypertension, which is a precursor for
heart disease [18, 19]. Inhibition of angiotensin-1-converting enzyme (ACE-I) activity has a potential link to a hypertension lowering effect, by preventing the conversion of
angiotensin I to aAngiotensin II, where the latter compound
is responsible for contracting the epithelial layer of arteries
causing an increase in blood pressure [20]. Previous studies have shown that peptides derived from soybean protein
with limited enzymatic hydrolysis possess ACE-I inhibitory
activity [21–23] and other bio-activities [24]. There have
been no studies determining the presence of higher than
normal amounts of methionine from high oleic acid soybean
meals, or the influence of amino acid content and sequence
which can elicit significant biological activities.
This is the first time that amino acid analysis of soybean lines with varying oleic acid composition and ACE-I
inhibitory activity assessment of alcalase enzyme-derived
peptides from their protein isolates has been studied. The
44 soybean lines used in this study were bred for attributes
including yield, protein content, low linoleic acid, high
oleic acid content, high yield and high protein. The major
objectives of this study were to analyze the amino acid
composition of the protein among 44 soybean lines, select
3 based on highest protein content and prepare protein
hydrolysates (peptide fractions) and test for ACE-I inhibitory activity.
Materials and Methods
The seeds of 44 soybean lines (R05-4509, R95-1705
(non-GMO), R05-4476, R05-4487, R05-4473, R05-4507,
13
R05-4492, Satellite, R05-4494, S03-543CR, N98-4445,
R05-5491, R05-5340, R05-4457, R05-4478, R05-4505,
R05-5362, Osage, S01-9265, UA-4805, 5601-T, R05-5351,
S04-4729RR, TN01-235, S04-3835 RR, R05-5510, Satellite,
N98-4445, Kristine, 5002-T, IA-3017, TN-5123, R05-4481,
R05-5342, R05-5494, CRR05-188, V01-1693, V01-6338,
V01-1702, S01-9364, Ozark, IA-2064, R05-5358, KS-5007)
from two Arkansas Agricultural Research Stations (ARS)
based in Fayetteville (FAY) and Stuttgart (STU) were provided by Dr. Pengyin Chen, Plant breeder and Professor,
Department of Crop Soil and Environmental Sciences, University of Arkansas. The Kjeltec 2200 auto-distillation unit
(Foss, Eden Prairie, MN, USA) was used to determine the
protein content in the flour. Rotovapor (Buchi, Flawil, Switzerland) was used for vacuum distillation and a Beckman
HPLC system (Fullerton, CA, USA) was used for quantitative amino acid analysis. An Ika mill (Ika-Werke, Staufen,
Germany) was used for grinding the samples. Food grade
enzyme alcalase 2.5L (EC 3.4.21.62) was purchased from
Novozyme (Bagsvaerd, Denmark) for preparing the protein
hydrolysates. All chemicals, solvents, and reagents with highest purity were purchased from Sigma (St. Louis, MO, USA).
Moisture and Protein Content Determination
The seeds were ground, passed through a 60-mesh sieve,
and the flour was collected, bagged and stored at 5 °C. The
moisture percentage of the flour was calculated based on
the AACC official method [25]. The soybean flour samples
were weighed in aluminum pans and dried at 135 °C for
3 h. The moisture percentage was calculated as the ratio
between the moisture lost from the sample and the actual
weight of the sample before drying. This was done in triplicate for all the 44 seed samples.
Protein content of the soybeans was performed using
the Kjeldahl method [26]. The soybean flour samples were
weighed in digestion tubes and digested for 1 h at 420 °C
after adding the Kjeldahl tablets and 10 mL sulfuric acid.
The samples were distilled and titrated against 0.1 N HCl
in the automated Kjeldahl distillation unit. The protein percentage was calculated with a conversion factor of 6.25 for
nitrogen. The moisture percentage was used to calculate the
protein content by dry weight for all the samples. All analyses were conducted in triplicate.
Determination of Amino Acid Composition
The amino acid analysis of the flour from the 44 lines was
conducted using the AOAC method [26]. The approximate
weight of each soybean meal test sample for amino acid
analysis was calculated by the formula: Ws = 1000/Ns;
where Ws is the weight of the sample in milligrams, and Ns
is the nitrogen content (%) in each sample.
J Am Oil Chem Soc
Performic acid solution was prepared and kept at room
temperature for 30 min and cooled in an ice bath for 15 min
before adding to the samples. All flour samples were
weighed into 250-mL Erlenmeyer flasks and cooled in an
ice bath. Five milliliters of performic acid was added to
each flask with the sample, stirred for 15 min, and all flasks
were kept in an ice bath for 16 h for oxidation. After oxidation, the performic acid was decomposed by adding 0.84 g
of sodium metabisulfite to each sample flask under a fume
hood and stirring for 10 min. The oxidized products were
hydrolyzed in 6 M HCl-phenol solution for 24 h at temperatures between 110 and 120 °C. The hydrolyzed sample
solution was cooled to room temperature, and 20 mL norleucine solution was added as HPLC elution standard. The
solutions were evaporated using a rotary evaporator under
vacuum (until 5–10 mL remained), diluted with sodium citrate buffer and the pH adjusted to 2.2. The volume of the
hydrolyzed sample solution was made up to 50 mL with
the buffer solution and stored at 5 °C in polyethylene bottles. The solutions were injected into a C18 ion exchange
column (heated to 70 °C) using an auto-sampler attached
to the HPLC system, and the amino acids were detected
based on the absorbance measured at 254 nm. Eluent buffer
solutions, procured from Pickering Laboratories (Mountain
View, CA, USA), containing sodium citrate and hydrochloric acid at varying pH (3.2, 4.2 and 6.4) were used. The
elution times of each amino acid on the column were compared to an amino acid standard and the amount of each
amino acid was calculated in mg/g based on the peak area.
Preparation of Protein Isolate and Enzymatic
Hydrolysis to Prepare Gastro-Intestinal Resistant
Peptide Fractions
hydrolysates. The hydrolysates were passed through each
column starting with 5 kDa as the permeates were collected and retentates were passed through 10 kDa and then
through a 50-kDa column following the similar process.
The specific peptide fractions, <5, 5–10 and 10–50 kDa,
were obtained as a results of ultrafiltration process, which
were freeze-dried and stored at 4 °C.
ACE-I Inhibitory Activity Assay
A modified method of Cushman and Cheung [28] was
used to conduct the ACE-I inhibition activity assay. Protein
hydrolysates at 500 µg/mL (30 µL), N-hippuryl-L-histidylL-leucine (HHL: 150 µL, 6.5 mM), and ACE-I (25 µL,
2.5 mU) were incubated at 37 °C for 1 h. Hydrochloric acid
(250 µL, 1 N) and ethyl acetate (1.5 mL) were added to
stop the reaction. The contents were mixed by vortexing
before centrifugation at 2000g for 5 min. One milliliter of
the top layer (containing hippuric acid extracted with ethyl
acetate) was collected, and ethyl acetate was removed using
a roto evaporator. The residual hippuric acid was dissolved
with deionized water (1 mL) and absorbance was measured
at 228 nm. Captopril (10 mM) was used as positive control while a solution containing ACE-I and HHL was used
as blank solution. Inhibition of ACE-I was calculated using
the formula:
% Inhibition = 1 − [(Absblank − Abstest )/Absblank ] × 100.
The % ACE-I inhibition was defined as the percentage of
ACE-I activity inhibited by a specific amount of peptides
and a dose response was conducted to determine the IC50
value for the peptide fractions with highest activity.
Statistical Analysis
Seeds from the three selected (based on high protein content) soybean lines (high oleic acid: N98-4445A, S03543CR; high yield: R95-1705) were ground, defatted and
passed through a 60-mesh (250 µm particle size) sieve.
Suspensions of the flours were prepared for solubilizing the
protein at an alkaline pH of 9.5. The protein solutions were
separated from the residue using a centrifuge (3000g for
15 min) and precipitated at isoelectric pH of 4.5 to prepare
the protein isolates. The protein isolate was digested using
the enzyme alcalase under optimized conditions to derive
varying size protein fragments or hydrolysates, as per the
previous study. The hydrolysis was conducted at pH 8.0
with 1.675 AU of enzyme incubated at 55 °C for 1 h for a
30 % degree of hydrolysis [24]. The hydrolysates were then
passed through a simulated gastro-intestinal (GI) environment using the enzymes, pepsin and pancreatin, at 37 °C
to derive GI-resistant protein hydrolysates [24, 27]. Ultrafiltration membrane columns with molecular cut-off—5,
10 and 50 kDa were used to separate the GI resistant
The JMP software from SAS Institute (Cary, NC, USA)
was used for the statistical analyses—Student’s t test, analysis of variance, means and standard deviations (P value
<0.05). The data were collected in triplicate for all the
experiments.
Results and Discussion
Moisture and Protein Content
The moisture percentage in the flour among the 44 soybean
lines ranged between 5.2 ± 0.0 and 12.9 ± 0.1 % (Table 1).
The moisture values varied due to the difference in the
agronomic growing conditions of the lines, moisture content at harvest and the processing conditions of the 44 lines.
Nevertheless, the moisture content did not have a significant impact on the protein content in the seeds.
13
J Am Oil Chem Soc
Table 1 Protein content (dry
and wet basis) of selected 44
soybean lines
Cultivar
Moisture %
Protein % (dry basis)
Yield attributesB
R05-4509 (STU)
R95-1705 (FAY)
R05-4476 (STU)
R05-4487 (STU)
R05-4473 (STU)
R05-4507 (STU)
R05-4492 (STU)
Satellite(STU)
R05-4494 (STU)
S03-543CR (FAY)
N98-4445A (FAY)
R05-5491 (STU)
R05-5340 (STU)
R05-4457 (STU)
R05-4478 (STU)
R05-4505 (STU)
R05-5362 (STU)
Osage (FAY)
S01-9265 (FAY)
UA-4805 (STU)
5601-T (STU)
R05-5351 (STU)
S04-4729RR (FAY)
TN01-235 (FAY)
S04-3835 RR (FAY)
R05-5510 (STU)
Satellite (FAY)
N98-4445A (Fdn)A
Kristine (FAY)
5002-T (STU)
IA-3017 (FAY)
TN-5123 (FAY)
R05-4481 (STU)
R05-5342 (STU)
R05-5494 (STU)
CRR05-188 (FAY)
V01-1693 (FAY)
V01-6338 (FAY)
V01-1702 (FAY)
S01-9364 (FAY)
Ozark (FAY)
IA-2064 (FAY)
R05-5358 (STU)
11.9 ± 0.0def
6.4 ± 0.0qrs
11.6 ± 0.0jkl
11.9 ± 0.0def
13.1 ± 0.1def
12.0 ± 0.0kl
11.5 ± 0.0efg
12.0 ± 0.0defg
11.8 ± 0.3efg
6.2 ± 0.2pqr
8.4 ± 0.1m
10.4 ± 0.1fghij
10.7 ± 0.4abc
5.3 ± 0.1rs
6.1 ± 0.1pqrs
11.4 ± 0.1a
10.8 ± 0.2ab
6.1 ± 0.1pq
9.4 ± 0.2kl
11.6 ± 0.0bcde
11.2 ± 0.0bcde
11.5 ± 2.1pqr
7.9 ± 0.3bcde
8.2 ± 0.3m
8.1 ± 0.1mn
9.9 ± 0.1cde
6.7 ± 0.2opq
6.2 ± 0.2pqr
6.2 ± 0.0abcd
11.7 ± 0.0efghi
7.9 ± 0.2hijk
7.6 ± 0.2mno
12.6 ± 0.1efg
12.9 ± 0.1ijk
11.8 ± 0.1ghij
6.4 ± 0.0pq
9.7 ± 0.1mn
5.7 ± 0.1qrs
10.3 ± 0.0mn
7.1 ± 0.1nop
8.3 ± 0.0m
5.2 ± 0.0s
9.7 ± 0.1efgh
53.5 ± 0.1a
52.7 ± 0.4ab
52.6 ± 0.1ab
52.3 ± 0.2ab
52.1 ± 0.2b
50.1 ± 0.1c
49.6 ± 0.6cd
48.9 ± 0.1de
48.6 ± 0.0def
48.4 ± 0.5defg
48.1 ± 0.4efg
47.6 ± 0.2efghi
47.4 ± 0.5fghij
47.4 ± 0.2fghij
47.4 ± 0.1fghij
47.3 ± 0.1ghijk
46.8 ± 0.2hijkl
46.7 ± 0.3ijkl
46.5 ± 0.2ijklm
46.4 ± 0.6ijklmn
46.4 ± 0.2jklmn
46.3 ± 0.8jklmno
46.1 ± 0.1klmno
46.0 ± 0.6lmno
46.0 ± 0.4lmno
45.7 ± 0.4lmnop
45.7 ± 0.3lmnop
45.6 ± 0.3lmnop
45.4 ± 0.8mnop
45.2 ± 0.3nop
45.2 ± 0.2nop
45.2 ± 0.3nop
45.2 ± 0.3nop
45.1 ± 0.4op
44.6 ± 0.3pq
44.5 ± 0.4pq
43.8 ± 1.4qr
43.7 ± 0.2qr
43.7 ± 0.6qr
43.5 ± 0.8qr
43.2 ± 0.3r
42.8 ± 0.2r
41.0 ± 0.2s
High protein and fatty acid
High yield
Fatty acid
Fatty acid
High protein and fatty acid
High protein and fatty acid
High protein and fatty acid
Fatty acid
High protein and fatty acid
High oleic acid
High oleic acid
Fatty acid
Fatty acid
High protein and fatty acid
High protein and fatty acid
High protein and fatty acid
Fatty acid
High yield
Low saturated fat
Check
Check
Fatty acid
Low linolenic acid
Low linolenic acid
Low linolenic acid
Fatty acid
Low saturated fat
Check
Low linolenic acid
Check
Low linolenic acid
High oleic acid
High protein and fatty acid
Fatty acid
Fatty acid
High oleic acid
Low linolenic acid
Low saturated fat
Low linolenic acid
Low linolenic acid
Conventional
Low linolenic acid
Fatty acid
KS-5007 (FAY)
10.5 ± 0.2l
40.8 ± 0.2s
Low linolenic acid
Values given are averages of three replications ± standard deviation and those connected by same letter in
each column are not significantly different
STU Stuttgart, FAY Fayetteville
A
N98-4445A (Fdn = foundation) is the check soybean line that is used as a positive control among high
oleic acid lines for testing the yield attributes
B
13
Yield attributes are the traits specific to the breed of the developed lines
J Am Oil Chem Soc
The protein percentage among all 44 lines ranged
between 40.8 ± 0.2 and 53.5 ± 0.1 % approximately by
dry weight. The differences in the protein content among
the soybean lines could be due to the genetic variation
among the soybean lines. There was a statistically significant difference in the protein content of the soybean
lines as shown in Table 1. Both Fayetteville and Stuttgart
Agricultural research stations produced high protein lines.
The variation in the protein content among the lines could
be attributed to the dissimilarities in the soils of the two
regions. Soybean lines R05-4509 (STU), R05-4476 (STU),
R05-4487 (STU), R05-4473 (STU), and R95-1705 had the
highest protein yields which are not significantly different
from each other according to the Student’s t test (P > 0.05)
while R05-4509 (STU) showed the highest protein content
of 53.5 ± 0.1 % among all the lines.
The highest protein content on dry basis (d. b.),
53.5 ± 0.1 % was from high protein line and fatty acid line,
R05-4509, which was not significantly different (statistically) from that of the high yielding line, R95-1705 with
52.7 ± 0.4 % protein. The high oleic acid line S03-543CR
had the highest protein content of 48.4 ± 0.5 % among
all high oleic acid soybean lines. The protein content of
R05-4509 (STU), R95-1705 R05-4476 (STU), R05-4487
(STU), and R05-4473 (STU) was much higher than normal and those with high oleic acid, S03-543CR and N984445A (Fay), are also found to have significantly high protein content. Lines grown for the high yield and high protein
attributes had higher protein content than the high oleic acid
lines which is consistent with previous studies [2]. Two
other high oleic acid lines, CRR05-188 and TN-5123 from
the Fayetteville ARS stations, had 45.2 and 44.5 % protein
which are not statistically different from each other and
similar to the ‘check’ lines—N98-4445A foundation and
5002-T. The ‘check’ soybean lines are used as positive control for comparison of an attribute (high yield, high protein
or high oleic acid, etc.) among the new breeds of soybean
seeds developed. ‘Foundation’ lines are those developed by
the breeder (copyrighted) for distribution among the growers. The lowest amount of protein on dry basis was found
in the KS-5007 low linolenic acid soybean which is 40.8 %.
Amino Acid Content
The amino acid analysis of the 44 soybean lines showed
variability in protein composition, while the presence of
high oleic acid content did not provide a wide variation.
Hence, other components, including isoflavones and oligosaccharides in the seed, could utilize the available carbon skeletons during development. A positive correlation
between protein, lipid, sugars and isoflavones during the
soy seed growth and maturity have been observed in previous studies [29]. The essential amino acid composition
of the seed protein in 44 lines is given in Table 2. The
amino acid composition among the high oleic acid soybeans lines did not differ significantly (P value <0.05, data
not included). High methionine and cysteine content were
observed in lines: R05-4494, R05-5491, 5002 T, Kristine, R05-5362 and R05-5352, which were 53.7 ± 1.4,
43.5 ± 0.1, 41.8 ± 4.8, 41.0 ± 0.8, 35.2 ± 3.6 and
39.9 ± 0.4 mg/g, respectively. Table 3 shows the list of
soybean lines which had the highest amounts of methionine content which can be sources of complete protein.
This could be due to the nitrogen assimilation during the
seed development which determines the amino acid composition of the seeds [16]. The methionine content in the
six soybean lines (addressed above) are significantly
higher in comparison to egg (34 mg/g) and milk (20 mg/g)
proteins (Table 4). Cysteine is the other sulfur containing amino acid which is non-essential to humans but is
required for the maintenance of protein structure and function. Table 3 shows the soybean lines which had the highest cysteine content: R05-4505—35.5 ± 0.3 mg/g, S019265—30.8 ± 4.8 mg/g, Satellite STU 23.7 ± 1.5 mg/g
and CRR05-188—25.5 ± 3.3 mg/g which is a high oleic
acid line. These values are higher than those found in milk
protein (8 mg/g) and are equal or higher in comparison to
egg protein (24 mg/g).
The CRR05-188 is the only high oleic soybean line that
showed higher cysteine content, although its seed protein
content is lower in comparison to S03-543CR and N984445A (high oleic acid lines). This indicates that sulfur
amino acid content in soybeans is not related to the amount
of total seed protein. The soybean line S03-543CR showed
the highest amounts of essential amino acids—threonine,
valine, isoleucine and leucine among all the lines tested
irrespective of their traits. It also showed significantly high
amounts of lysine amino acid when compared with the 44
soybean lines. This shows that soybean lines bred for high
oleic acid can also provide substantial essential amino acid
content along with high protein. The protein content in the
TN-5123 high oleic acid soybean line was not significantly
different in comparison to CRR05-188, but the essential
amino acid content was lower in comparison to other high
oleic acid lines. Hence, other factors, including soil environment and growth conditions, affect the protein formation during seed development.
Among all the soybean lines, the compositions of glutamine, asparagine and lysine amino acids are highest in
that order, respectively (Fig. 1). Glutamine is essential for
gut health while asparagine maintains the integrity of the
nervous system [30, 31]. Lysine is known to be an essential
amino acid which helps in serotonin regulation and has a
moderating effect on blood pressure and the incidence of
stroke [31, 32]. The amino acid analysis procedure from
AOAC official methods (994.12) was selected to quantify
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J Am Oil Chem Soc
Table 2 Essential amino acid composition (mg/g protein) in 44 soybean lines grown in the state of Arkansas
Amino acid
R05-4509
R95-1705
R05-4476
R05-4487
R05-4473
R05-4507
Cysteine
Threonine
Valine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Histidine
15.2 ± 0.0ab
44.1 ± 0.7mn
31.4 ± 0.9
32.7 ± 0.8jk
67.4 ± 0.7k
109.0 ± 1.0q
22.3 ± 1.2f
40.5 ± 0.9j
53.8 ± 0.6jk
14.8 ± 0.4a
41.8 ± 0.6l
40.7 ± 0.5l
40.2 ± 0.3p
68.9 ± 0.5l
113.5 ± 0.9s
24.3 ± 0.9g
46.7 ± 0.6m
56.3 ± 0.4l
15.1 ± 0.5ab
43.7 ± 0.7m
35.5 ± 0.8ij
37.1 ± 0.8n
67.4 ± 0.6k
112.0 ± 0.9rs
23.5 ± 1.0g
45.1 ± 0.8lm
54.7 ± 0.6k
14.5 ± 0.7a
42.2 ± 0.5lm
29.5 ± 0.6g
32.0 ± 0.5jk
64.2 ± 0.6i
107.3 ± 1.3p
20.6 ± 1.1ef
46.0 ± 0.9m
51.6 ± 0.5i
13.4 ± 0.8a
36.8 ± 0.5k
23.1 ± 0.6cd
22.3 ± 0.5e
55.6 ± 0.5de
101.6 ± 0.9m
19.1 ± 0.7e
41.4 ± 0.6k
44.9 ± 0.5g
14.6 ± 0.3a
41.6 ± 0.6l
38.2 ± 0.5k
36.2 ± 0.4m
64.5 ± 0.3i
109.5 ± 0.7q
21.4 ± 0.9f
45.4 ± 0.4lm
52.7 ± 0.4j
Amino acid
R05-4492
Sat (STU)
R05-4494
S03-543CR
N98-4445A
R05-5491
Cysteine
Threonine
Valine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Histidine
16.5 ± 0.0b
21.0 ± 0.0ef
29.2 ± 0.1g
24.3 ± 0.1f
66.7 ± 0.4jk
109.7 ± 1.5q
26.4 ± 0.3h
47.1 ± 0.1n
52.5 ± 0.1j
23.7 ± 1.5e
21.8 ± 0.0ef
22.7 ± 0.1c
22.0 ± 0.0e
57.8 ± 0.4ef
60.7 ± 0.3g
17.1 ± 0.1d
35.4 ± 0.1fg
33.6 ± 0.0cd
15.9 ± 0.1ab
20.0 ± 0.1de
25.6 ± 0.6e
22.5 ± 0.0e
68.3 ± 0.2kl
98.1 ± 6.5k
53.7 ± 1.4p
31.1 ± 2.7d
49.3 ± 0.4h
14.2 ± 0.5a
45.2 ± 0.7n
42.1 ± 0.5m
39.2 ± 0.6o
69.1 ± 0.5l
123.8 ± 0.8u
24.3 ± 1.2g
48.0 ± 0.4o
56.2 ± 0.5l
14.9 ± 0.3a
18.5 ± 0.6d
27.9 ± 0.4f
25.9 ± 0.3f
51.3 ± 0.5b
53.4 ± 0.7de
14.2 ± 0.0bc
30.1 ± 0.2c
30.9 ± 0.2b
15.5 ± 0.6ab
21.3 ± 0.4ef
33.8 ± 1.4i
31.6 ± 1.7j
71.2 ± 3.9m
117.2 ± 7.0t
43.5 ± 0.1o
46.1 ± 3.0m
56.6 ± 2.5l
Amino acid
R05-5340
R05-4457
R05-4478
R05-4505
R05-5362
Osage
Cysteine
Threonine
Valine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Histidine
14.6 ± 0.6a
19.4 ± 0.3d
35.0 ± 1.0ij
31.8 ± 0.3j
69.6 ± 0.5l
104.2 ± 0.9o
33.6 ± 1.3j
47.6 ± 1.6n
55.1 ± 0.6l
21.3 ± 2.3cd
21.6 ± 0.0ef
23.8 ± 0.0cd
23.6 ± 0.0ef
58.1 ± 0.2f
60.0 ± 0.1g
14.2 ± 0.0bc
35.0 ± 0.2fg
33.9 ± 0.1cd
15.3 ± 0.1a
19.9 ± 0.2d
26.5 ± 0.4ef
22.9 ± 0.0ef
65.4 ± 0.3ij
107.5 ± 0.9p
33.3 ± 4.3k
44.5 ± 0.3l
52.7 ± 0.3j
35.1 ± 0.3h
21.2 ± 0.0ef
20.2 ± 0.1bc
19.6 ± 0.1d
56.6 ± 0.4e
60.2 ± 1.0g
13.8 ± 0.1b
34.7 ± 0.2f
32.2 ± 0.2c
14.9 ± 0.2a
18.6 ± 0.8d
36.4 ± 0.8j
31.3 ± 0.2j
68.3 ± 0.6kl
103.3 ± 0.1mn
35.2 ± 3.6l
47.4 ± 0.9n
54.9 ± 0.2kl
14.7 ± 0.4a
41.4 ± 0.7l
24.7 ± 0.9d
24.2 ± 0.7f
60.4 ± 0.5fg
104.1 ± 1.1o
19.9 ± 0.5e
44.1 ± 0.8l
51.5 ± 0.6i
Amino acid
S01-9265
UA-4805
5601T
R05-5351
S04-4729RR
Cysteine
Threonine
Valine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Histidine
Amino acid
Cysteine
Threonine
Valine
Isoleucine
Leucine
Lysine
13
g
30.8 ± 4.8
21.9 ± 0.0ef
22.7 ± 0.0c
22.3 ± 0.1e
57.7 ± 0.3ef
61.8 ± 0.0h
16.0 ± 0.8d
34.1 ± 0.1f
34.5 ± 0.0d
S04-3835RR
ab
15.7 ± 0.2
20.8 ± 1.4de
36.4 ± 0.4j
34.6 ± 0.2l
56.0 ± 1.5e
54.3 ± 0.0c
a
13.1 ± 0.6
16.7 ± 0.6c
29.6 ± 0.7g
27.0 ± 0.1g
62.3 ± 0.5gh
96.1 ± 0.2j
34.5 ± 3.8l
41.3 ± 1.3k
50.0 ± 0.4hi
R05-5510
a
14.3 ± 0.6
14.9 ± 0.3b
24.9 ± 1.2de
17.2 ± 0.1bc
57.6 ± 0.5ef
102.9 ± 0.3m
33.0 ± 3.2j
44.6 ± 1.4l
45.3 ± 0.7g
Sat (Fay)
b
16.6 ± 0.0
19.5 ± 1.3d
32.6 ± 0.0h
30.9 ± 0.0i
71.6 ± 0.2m
111.0 ± 1.4r
b
16.4 ± 0.1
20.0 ± 1.6de
31.6 ± 0.5h
29.7 ± 0.4i
53.3 ± 0.8c
50.1 ± 0.0c
13.7 ± 0.2b
32.0 ± 0.6de
32.8 ± 0.2c
N98 (Fdn)
de
22.0 ± 1.9
21.1 ± 0.0ef
19.1 ± 0.0b
16.9 ± 0.1b
54.0 ± 0.0d
63.9 ± 0.4i
c
19.2 ± 1.2
27.0 ± 1.7h
30.0 ± 3.6gh
31.4 ± 4.2j
65.8 ± 3.6ij
56.0 ± 3.0f
28.5 ± 0.7ij
33.4 ± 2.9e
38.3 ± 2.3f
Kristine
a
14.0 ± 05
43.5 ± 0.6m
35.4 ± 0.8ij
34.4 ± 0.8l
62.8 ± 0.8h
109.6 ± 1.4q
TN01-235
15.4 ± 0.2ab
13.7 ± 0.1a
19.7 ± 0.9b
14.6 ± 0.0a
43.5 ± 0.1a
48.3 ± 0.1bc
12.4 ± 0.0a
26.6 ± 0.0b
27.3 ± 0.0a
5002T
b
16.2 ± 0.7
21.7 ± 0.7ef
30.6 ± 1.6gh
27.6 ± 1.6g
68.6 ± 4.0kl
102.9 ± 0.0m
14.5 ± 0.2a
16.8 ± 1.3c
22.0 ± 0.4c
19.5 ± 1.2d
67.1 ± 0.1k
103.0 ± 0.7mn
J Am Oil Chem Soc
Table 2 continued
Amino acid
S04-3835RR
b
R05-5510
Sat (Fay)
j
32.5 ± 1.9
50.2 ± 1.5p
58.7 ± 0.7m
N98 (Fdn)
d
18.0 ± 0.0
33.7 ± 0.0e
32.3 ± 0.0c
Kristine
g
23.4 ± 0.9
45.3 ± 1.0lm
52.0 ± 0.6j
5002T
n
41.8 ± 4.8n
38.6 ± 0.2i
54.5 ± 2.1k
41.0 ± 0.9
39.0 ± 0.1i
53.9 ± 1.9jk
Methionine
Phenylalanine
Histidine
13.7 ± 0.7
36.4 ± 0.1h
34.4 ± 0.2d
Amino acid
IA-3017
TN-5123
R05-4481
R05-5342
R05-5494
CRR05-188
Cysteine
Threonine
Valine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Histidine
20.1 ± 0.4c
24.8 ± 0.6g
35.8 ± 0.8ij
35.6 ± 0.1lm
66.1 ± 0.7jk
53.7 ± 2.1de
20.2 ± 1.0ef
37.0 ± 0.4h
38.2 ± 0.5f
15.0 ± 0.1ab
20.0 ± 0.7de
16.8 ± 0.0a
16.7 ± 0.8b
51.9 ± 0.8b
61.0 ± 1.7h
16.7 ± 1.9d
24.9 ± 0.9a
30.0 ± 1.1b
19.7 ± 0.1c
24.0 ± 1.2g
25.9 ± 0.4e
22.7 ± 0.0e
57.9 ± 0.0ef
62.1 ± 0.4h
14.2 ± 1.2bc
34.5 ± 1.0f
33.1 ± 0.2cd
16.7 ± 0.0b
24.2 ± 0.1g
27.0 ± 0.2f
28.0 ± 0.0gh
60.8 ± 0.0fg
50.5 ± 1.7c
20.2 ± 2.5ef
29.3 ± 0.7c
34.2 ± 0.0d
16.6 ± 0.2b
23.5 ± 0.2fg
26.5 ± 2.3ef
27.4 ± 2.5g
57.5 ± 0.9ef
52.8 ± 0.5d
26.4 ± 2.0h
29.1 ± 1.2c
32.7 ± 0.7c
25.5 ± 3.3f
19.8 ± 0.0d
31.7 ± 0.0h
32.3 ± 0.1jk
66.4 ± 0.2jk
61.8 ± 0.5h
15.8 ± 0.1c
38.7 ± 0.3i
38.5 ± 0.0f
Amino acid
V01-1693
V01-6338
V01-1702
S01-9364
Ozark
IA 2604
c
b
Cysteine
Threonine
Valine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Histidine
20.3 ± 0.4
22.0 ± 0.9f
26.9 ± 0.5ef
25.4 ± 0.1f
61.1 ± 0.6g
60.9 ± 0.8g
21.4 ± 0.4f
33.1 ± 1.5e
35.3 ± 0.3d
16.4 ± 0.1
17.5 ± 1.5cd
23.1 ± 1.4cd
17.3 ± 1.1bc
53.1 ± 0.3c
49.1 ± 0.4b
20.9 ± 2.0ef
26.6 ± 0.2b
30.1 ± 0.6b
Amino acid
R05-5358
KS-5007
Cysteine
Threonine
Valine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Histidine
b
16.3 ± 0.2
19.7 ± 0.6d
21.8 ± 0.9c
19.2 ± 1.0d
73.3 ± 1.1n
137.8 ± 3.0v
39.9 ± 0.4m
36.5 ± 1.2h
16.4 ± 0.1b
34.2 ± 0.2j
32.1 ± 0.1h
27.2 ± 0.1g
63.8 ± 0.2i
96.9 ± 0.3j
26.1 ± 0.1h
44.2 ± 0.0l
50.7 ± 1.5hi
54.2 ± 0.1k
a
14.7 ± 0.3
21.6 ± 0.9ef
24.2 ± 1.0de
32.3 ± 1.7jk
58.9 ± 0.2f
46.8 ± 1.3a
27.8 ± 0.3hi
26.8 ± 0.6b
31.4 ± 1.0b
b
16.7 ± 0.2
19.8 ± 0.0d
30.6 ± 1.7gh
27.7 ± 1.6g
65.6 ± 0.7j
111.8 ± 2.7r
32.6 ± 1.5j
41.2 ± 2.9k
55.9 ± 1.1l
b
16.0 ± 0.5
44.2 ± 0.7mn
32.2 ± 0.9h
31.3 ± 0.8j
64.5 ± 1.0i
100.1 ± 1.3l
20.3 ± 0.6ef
48.5 ± 0.9o
54.2 ± 0.7k
20.6 ± 0.1c
22.9 ± 1.5f
31.4 ± 0.2h
30.7 ± 0.1i
59.5 ± 0.1f
54.5 ± 0.1e
14.0 ± 0.4bc
33.1 ± 0.8e
36.3 ± 0.1e
Values are mean ± standard deviation of triplicate analysis and those connected by same letter in each row are not significantly different from
each other (P < 0.05)
Soybean lines: Sat (Fay) Satellite (Fayetteville); Sat (STU) Satellite (Stuttgart); N98 (Fdn) N98-4445A (Foundation)
the sulfur-containing amino acids methionine and cysteine.
However, quantification of aromatic amino acids, tryptophan and tyrosine, was affected during hydrolysis and oxidation and these were not detected during the ion-exchange
liquid chromatography.
All 44 soybean lines showed high amounts of lysine
which agrees with the accepted notion that legume seeds
are rich in this amino acid [33]. While glutamine and asparagine were non-essential amino acids, supplementation of
lysine is essential to humans since it is not synthesized in
the body. Lysine plays an important role in transamination
reactions and is utilized to produce vital proteins, including elastin and collagen [34–36]. The comparison of essential amino acids (range) among the tested 44 soybeans and
other protein sources including eggs and milk are given in
Table 4.
Researchers have engineered quality traits through plant
breeding to enhance the sulfur amino acid content, even
though natural mutations in soybeans have also expressed
these beneficial effects. The genetic alterations have been
13
J Am Oil Chem Soc
Table 3 Soybean lines
with comparatively higher
methionine and cysteine content
(mg/g of protein) among the
tested soybean lines
Soybean line
Methionine (mg/g)
a
Yield attribute
R05-4494
R05-5491
5002-T
Kristine
R05-5362
R05-5358
UA-4805
R05-4478
53.7 ± 1.4
43.5 ± 0.1b
41.8 ± 4.8bc
41.0 ± 0.8bc
35.2 ± 3.6d
39.9 ± 0.4bc
34.5 ± 3.8d
33.3 ± 4.3d
High protein and fatty acid
Fatty acid
Check
Low linolenic acid
Fatty acid
Fatty acid
Check
High protein and fatty acid
Soybean line
Cysteine (mg/g)
Genetic attribute
R05-4505
S01-9265
Satellite STU
35.1 ± 0.3a
30.8 ± 4.8a
23.7 ± 1.5b
High protein and fatty acid
Low saturated fat
Fatty acid
CRR05-188
25.5 ± 3.3ab
High oleic acid
Values are mean ± standard deviation and those connected with same letter are not significantly different
(P value >0.05)
STU Stuttgart
Table 4 Comparative analysis of essential amino acid composition
(mg/g protein) of egg, milk and soy protein
Amino acid
Egg proteina
Milk proteina
Soy proteinb
Cysteine
Threonine
Valine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
24
47.8
48.2
51.4
82.9
66.4
34.9
59.5
7.9
47.3
63.8
68.5
103.5
68.3
20.2
46.7
13.1–35.1
14.9–45.2
16.8–42.1
14.6–40.2
43.5–73.3
46.8–137.8
12.4–53.7
24.9–50.2
Histidine
23.5
21.8
27.3–58.7
a
Source: FAO. Accessed at: www.fao.org/docrep
b
Values for soy protein are from the 44 soybean lines that are presented as a range
tested specific to the trait loci that trigger the formation of
methionine and cysteine during the seed growth [17, 37].
Soil sulfur and nitrogen content during the growth of the
soybeans also affects the methionine and cysteine content
in the soybean protein [38]. Other genetic attributes like
high protein, high monounsaturated fatty acid content,
and higher yields or disease resistance may contribute to
enhanced sulfur amino acids, but this is inconclusive.
Activity of Protein Hydrolysates
Three soybean lines, S03-543CR and N98-4445A (with
high oleic acid) and R95-1705 (high yield and non-GMO),
were selected based on their high protein content. The
13
alkali extraction method provided >90 % protein yield in
the isolates (yield: >83 % d. b. by mass balance) which
were used to prepare the protein fragments. The optimal
conditions to accomplish the alcalase enzymatic hydrolysis
of the proteins in order to derive peptides of varying sizes
were achieved using a statistical design for a 30 % degree
of hydrolysis to derive protein hydrolysates or peptides of
varying sizes [24]. The ultrafiltration of GI-resistant protein hydrolysates using molecular cut-offs columns—5, 10
and 50 kDa provided the fractions <5, 5–10 and 10–50 kDa
(yield: 1.8–2.1 % d. b. by mass balance, derived from the
isolate) for each soybean line with protein content ranging
between 89 and 92 %. These peptide fractions were tested
for ACE-I inhibitory activity.
The nine fractions (3 fractions from 3 soybean lines)
obtained from enzymatic hydrolysis of the protein from
three soybean lines were tested for ACE-I inhibitory property at a concentration of 500 µg/mL. The results showed
low activity in comparison to the positive control, captopril
(approximately 75 % inhibition). The highest inhibition
among the fractions was 48.9 ± 4.0 % by the 5–10 kDa
obtained from R95-1705 soybean line (Fig. 2), and this
fraction was chosen for the dose response study to determine the minimum inhibitory concentration. The <5-kDa
fraction from the high oleic acid soybean line N98-4445A
showed an inhibition of 42.2 ± 1.3 % which was the
only other significant activity observed against the ACE-I
enzyme among all the protein hydrolysate fractions. Peptides of both large and small sizes have shown bioactivities
in previous studies and have exhibited functionalities that
can be used in food products [21, 39]. The hydrolysates
tested for GI resistance could potentially be available for
J Am Oil Chem Soc
Fig. 1 One-way analysis of amino acid profile among the 44 soybean
lines. The mean weight (mg/g of protein) from amino acid analysis
are shown for each amino acid detected in the protein
absorption through the intestine when consumed as food
and elicit the health beneficial bioactivities in the target
tissues. Researchers have shown that peptides of various
molecular sizes are absorbed through the intestinal wall
but the ability of absorption decreases with an increase in
molecular size [40].
A dose response study of the 5- to 10-kDa fraction (R951705) revealed an increase in ACE-I inhibitory activity as
the dosage of the fractions increased from 200 to 1200 µg/
mL (data not shown). The highest inhibition was achieved
at 1200 µg/mL concentration (75.5 ± 2.8 %) which was
not significantly different from that shown by 1000 µg/mL
concentration (72.4 ± 1.4 %). An increase in ACE-I inhibition was observed at ≥800 µg/mL concentration, although
the highest inhibition by the fractions was significantly
lower in comparison to the positive control. The dose
response provided the inhibitory concentration at 50 %
activity (IC50) of the 5- to 10-kDa protein fraction from
R95-1705 to be 563 µg/mL. These results are significant
as R95-1705 is a non-GM soybean line, which can have a
potential impact on its utilization in foods or supplemental
therapeutics. The ACE-I inhibitory activity can be attributed to the pool of peptides from 5–10 kDa and their amino
acid sequences. However, other studies have reported significant ACE-I inhibitory activity by similar molecular size
peptide fractions which were derived from marine protein
sources [41]. Previous studies from the current research
group have shown the ability of large molecular size protein hydrolysates (>50 kDa) to have ACE-I inhibition [42].
Researchers have also reported ACE-I and atherosclerosis
inhibition by hydrolysates obtained from both glycinin
and β-conglycinin fractions of soy protein [43, 44]. Hence,
the peptide fraction from R95-1705 (5–10 kDa) with antiACE-I activity may have been derived from either the glycinin (11S) or β-conglycinin (7S) fractions of the proteins
which needs further examination.
Several factors can be attributed to the substantial
anti-ACE-I activity that can be studied, starting with the
purification of the peptide pool from the 5- to 10-kDa
fractions. Amino acid composition of the fractions, specifically of the peptides which elicit the activity, will reveal
their chemical nature and provide an explanation which
needs to be investigated. Studies have also shown that
protein hydrolysates and peptides derived from various
Fig. 2 ACE-I inhibitory activity of soybean protein hydrolysates at 500 µg/mL concentration. Captopril—10 mM concentration. Bars represented by same letter are not significantly different (P < 0.05). Values are mean ± standard deviation
13
J Am Oil Chem Soc
food sources possess anti-ACE-I activities [41, 42, 45].
Although most researchers tested (pure) peptides derived
from either fermentation or enzymatic hydrolysis, very
few studies have demonstrated the bioactivity of GI-resistant protein hydrolysates which contain a pool of peptides.
Preparation of hydrolysates is economical in comparison
to derivation of pure peptides. Protein hydrolysates can
provide synergistic effects and have shown multiple bioactivities [24, 27].
Conclusion
The protein contents of soybean lines R05-4509, R951705, R05-4476 and R05-4487 were higher among all the
lines tested. Soybean lines with ‘high oleic acid’ (S03543CR and N98-4445A) were also found to have substantially enhanced protein content (approximately 48 % d. b.).
Lines grown for the ‘high protein and fatty acid’ attribute
had 47–52 % (d. b.) protein content which was comparable
to that in the ‘high oleic acid’ lines. Amino acid analysis
showed a significantly higher methionine levels (P value
<0.05) in soybean lines recognized for protein and fatty
acid content (R05-4494 and R05-4478). Both methionine
and cysteine contents were elevated in the soybean lines
attributed for protein and fatty acid.
The protein fraction from the R95-1705, a non-GM soybean, showed highest ACE-I inhibition. This is the first
time ACE-I inhibition has been achieved with GI-resistant
non-GMO soy protein hydrolysates derived by enzymatic
digestion of high purity protein isolates. The 5- to 10-kDa
protein fraction at a higher dose presented an enhanced
ACE-I inhibitory activity. In conclusion, this study provides
amino acid composition of soybean lines that are grown
for definite yield attributes and demonstrates functional
activity of protein/peptide fractions derived from selected
soybean lines. The impact of ACE-I inhibition by protein
hydrolysates from the R95-1705 soybean line is significant
as it is a non-GMO soybean line.
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