Composition and bioactive factor content (1)
Journal of the Science of Food and Agriculture
J Sci Food Agric 87:112–119 (2007)
Composition and bioactive factor
content of cowpea (Vigna unguiculata L.
Walp) raw meal and protein concentrate
Leticia Olivera-Castillo,1∗ Fabiola Pereira-Pacheco,2 Erik Polanco-Lugo,2
Miguel Olvera-Novoa,1 Jose´ Rivas-Burgos3 and George Grant4
1 Centro
´ y de Estudios Avanzados del IPN, Unidad Merida, Antigua Carretera a Progreso Km 6, 97310 Merida, Yucatan,
de Investigacion
Mexico
2 Facultad de Ingenieria Qu´ımica, Universidad Autonoma
´
´
de Yucatan, Av. Juarez
No 421, Cd. Industrial, 97288 Merida, Yucatan, Mexico
3 Instituto Tecnologico de Merida, Antigua Carretera Progreso Km 5, 97118 Merida, Yucatan, Mexico
4 The Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK
Abstract: Analysis was done of the composition and bioactive factor content of whole meal, processed meal and
protein concentrate from a cowpea cultivar (Vigna unguiculata L. Walp var. IT86D-719) grown in Yucatan, Mexico
and of changes in these parameters after soaking and dehulling. Both meals had a high protein content (247.53 and
257 g kg−1 dry matter (DM) respectively). The protein concentrate was rich in protein (786 g kg−1 DM) and lipids
(58.47 g kg−1 DM) and had an amino acid profile similar to that of the processed meal. The amino acid profiles
of the meals almost covered human dietary requirements based on FAO/WHO/UNU-suggested profiles but were
deficient in sulphur amino acids. Trypsin inhibitor activity was high in both meals compared with levels found in
previous studies. Trypsin inhibitor activity in the concentrate was not eliminated but was significantly reduced.
Lectin activity, tannin levels, phytate levels and α-amylase inhibitor activity were relatively low in the meals, and
cyanogenic glucosides were not detected. Residual amounts of α-amylase inhibitors, tannins and phytate were
observed in the concentrate, and lectin activity was not detected. Results indicate that V. unguiculata L. Walp var.
IT86D-719 meals and protein concentrate are good potential foodstuffs in the Yucatan region.
2006 Society of Chemical Industry
Keywords: Vigna unguiculata L. Walp; proximate analysis; amino acid composition; antinutritional factors;
Yucatan, Mexico
INTRODUCTION
Legume grains are a primary source of dietary
protein in many regions of the world.1 However,
yields vary greatly according to plant species,
environmental conditions and horticultural practices.
The actual nutritional value of these crops is also
compromised by an apparently low digestibility of
their constituent proteins and by adverse effects of
seed components on body metabolism, particularly
bioactive/antinutritional factors such as lectins, trypsin
inhibitors, tannins, etc.1,2
Most legume seeds therefore require extensive
processing before they can be used safely and
effectively. Various methods have been developed
to improve the nutritional properties of legume
seed products, including sprouting,3 soaking and
cooking,4 extrusion5 and isolation of the major storage
(globulin-like) proteins, i.e. protein concentration.
Protein concentrate quality may vary, however,
depending on the exact preparation conditions,
and thus new legume protein concentrates require
chemical characterisation of their composition and
bioactive/antinutritional factor content. Despite this
variation, protein concentrates generally have good
nutritional quality, partially because globulin-like
proteins have low antinutritional factor levels.6
Cowpeas (Vigna unguiculata) grow well in a diverse
range of conditions and environments and contain only
moderate levels of bioactive/antinutritional factors.7
The National Institute of Forestry, Agricultural
and Livestock Research (Instituto Nacional de
Investigaciones Forestales, Agr´ıcolas y Pecuarias
(INIFAP)) located in Uxmal, Yucatan, Mexico
recently identified V. unguiculata L. Walp var. IT86D719, a variety created at the International Institute
for Tropical Agriculture (IITA) in Ibadan, Nigeria, as
having good agronomic potential for use as a crop
plant in the Yucatan Peninsula region. Indeed, it
was found to have a high protein and starch content
and a yield of up to 3.5 T ha−1 , significantly higher
∗
´ y de Estudios Avanzados del IPN, Unidad Merida, Antigua Carretera a Progreso Km 6,
Correspondence to: Leticia Olivera-Castillo, Centro de Investigacion
97310 Merida, Yucatan, Mexico
E-mail: olivera@mda.cinvestav.mx
Contract/grant sponsor: British Council/Mexico HEL Program; contract/grant number: MXC/991/83
Contract/grant sponsor: Scottish Executive Environment and Rural Affairs Department
Contract/grant sponsor: International Foundation for Science; contract/grant number: E/3171-1
(Received 25 November 2004; revised version received 7 October 2005; accepted 15 June 2006)
Published online 16 October 2006; DOI: 10.1002/jsfa.2684
2006 Society of Chemical Industry. J Sci Food Agric 0022–5142/2006/$30.00
Bioactive factor content of cowpea raw meal and protein concentrate
than that achieved with other legumes grown in the
region. Despite its promise, usage of cowpea by the
regional population remains low; unfamiliarity with
this legume, its horticulture and possible uses means
that there is only a limited local market for it.
As part of a developmental programme, V.
unguiculata L. Walp var. IT86D-719 seeds were
provided to small-scale farmers in rural Yucatan
and grown in accordance with local horticultural
practices. The composition, amino acid profile and
bioactive/antinutritional factor content of whole meal,
processed meal and protein concentrate prepared from
this legume are reported in this study. Additional
information is provided on changes in the proximal
and antinutritional composition resulting from the
protein concentration process.
EXPERIMENTAL
Seed preparation
Vigna unguiculata L. Walp (IT86D-719) seeds
from INIFAP were cultivated in the rural area of
Santa Elena, Yucatan, harvested when mature and
dry, cleaned and stored at 4 ◦ C until processing.
Representative samples of the seeds were ground in
a hammer mill fitted with a 1 mm2 mesh. Whole V.
unguiculata seeds (3 kg) were soaked (1:4 w/v) for 16 h
at 25 ◦ C in sodium bisulphite (2 g L−1 ) solution. The
seeds were then cracked in a manual mill and the
hulls separated off by water flotation (dehulling). The
cotyledons were cleaned and then dried in a lyophiliser,
though one sample of fresh cotyledons was dried in
a convection oven at 40 ◦ C to determine if dry heat
reduced trypsin inhibitor levels.
Protein concentrate preparation
Protein extraction was done using a modified version
of the isoelectric method.8 Briefly, the cotyledons were
passed through an industrial mill (Koch mod. C352Z,
Kansas City, MO, USA) to produce a homogenous
dough, which was then ground (1:3 w/v) in water in
a colloidal mill (Micron Mod. WB-1, M´exico, DF,
Mexico). The pH of the resulting slurry was adjusted
to 9 using 0.05 mol L−1 NaOH. After approximately
20 min the suspension was filtered through 0.150
and 0.106 mm2 meshes to remove fibrous material.
The remaining slurry was stored without agitation
at 4 ◦ C for 12 h to allow the starch to settle. The
supernatant was decanted and its pH adjusted to 4.33
with 0.5 mol L−1 HCl. It was then heated at 80 ◦ C for
10 min and stored for 12 h at 4 ◦ C. The precipitated
globulin-enriched fraction (i.e. protein concentrate)
was recovered by centrifugation (1500 × g, 12 min)
and subsequent drying in a convection oven at 40 ◦ C
for 24 h.
Proximate composition
Moisture, ash, crude fibre and crude fat contents
in the whole meal, processed meal and protein
concentrate were determined according to standard
J Sci Food Agric 87:112–119 (2007)
DOI: 10.1002/jsfa
methods.9 Nitrogen content was estimated by gas
chromatography using a ThermoQuest NCS analyser
(ThermoQuest Flash EA 1112, Rodano, Italy). Crude
protein content was calculated as N × 6.25. All
analyses were run in triplicate.
Amino acid analysis
Amino acid analysis of the defatted meals and
defatted protein concentrate was done using a
four-step Pico-Tag method.10 In the Pico-Tag
station, hydrolysis was done using 3 mol L−1
HCl under vacuum at 104 ◦ C for 24 h, followed by redrying with ethanol/water/triethylamine
(2:2:1 v/v/v) solution and derivatisation with
ethanol/triethylamine/water/phenylisothiocyanate
(7:1:1:1 v/v/v/v) reagent. Once derivitised, aliquots
were subjected to reverse phase liquid chromatography
in a Waters high-performance liquid chromatography
(HPLC) system (Waters Corporation, Milford, MA,
USA). Tryptophan was determined by a colorimetric
method.11 All analyses were run in triplicate.
Analysis of antinutritional factors in meals and
protein concentrate
Trypsin inhibitor activity, α-amylase inhibitor content,
lectin activity, tannic acid levels, phytate content and
cyanogenic glucoside levels were determined in the
whole meal (WM), processed meal (PM) and protein
concentrate (PC) as follows.
Trypsin inhibitor activity was assayed by the method
of Liu and Markarkis12 using benzoyl-DL-arginine-pnitroanilide (BAPNA) as substrate and porcine trypsin
(Type II-S, Sigma Chemical St. Louis, Mo, USA).
One trypsin unit (TU) was defined as 0.01 at A410
under the assay conditions, and trypsin inhibitor
activity was expressed as trypsin units inhibited (TUI)
kg−1 dry matter (DM) and kg−1 protein. Trypsin
inhibitor activity in the WM, PM and PC was also
determined from diluted samples of the fractions with
40–60% inhibition of enzyme activity and expressed
as g enzyme inhibited kg−1 DM.
α-Amylase inhibitor content was determined by
the procedure of Piergiovanni13 and expressed as g
amylase inhibited kg−1 DM and kg−1 protein.
Lectin activity (haemagglutination) was assayed by
the serial dilution method of Armour et al.14 using
human, bovine and hamster red blood cells previously
treated with trypsin. One haemagglutinating activity
unit (HU) was defined as that contained in the amount
of sample in the final dilution which caused 50%
agglutination of the blood cells. Lectin activity was
also determined using pure Phaseolus vulgaris lectin
(Sigma Chemical) as a standard and expressed as g
lectin equivalent kg−1 DM.
Tannic acid levels were determined using a
spectrophotometric method15 and expressed as g
tannic acid kg−1 DM and kg−1 protein.
Phytate content was determined by an anion
exchange method.9 Phytate was extracted from
triplicate dry samples using diluted HCl (24 g L−1 ).
113
L Olivera-Castillo et al.
The extract was then mixed with ethylene diamine
tetraacetic acid (EDTA)/NaOH solution and placed
in an ion exchange column. The phytate was eluted
with 0.7 mol L−1 NaCl solution and wet digested
with HNO3 /H2 SO4 mixture to release phosphorus,
which was measured calorimetrically at 640 nm and
expressed as g phytic acid kg−1 DM and kg−1 protein.
Cyanogenic glucoside levels were determined using
the method of Lucas and Sotelo,16 based on the
specific Guinard reaction, and expressed as g HCN
kg−1 DM.
Statistical analysis
With the exception of lectin activity, results were
subjected to one-way analysis of variance (ANOVA) at
P < 0.05. Differences between means were evaluated
by the Newman–Keuls test; normality was tested
using the Wilk–Shapiro test. When necessary, values
were normalised using natural logarithms. Lectin
activity results were compared using the Student t
test (P < 0.05). Means for all data were calculated
from three replicates (n = 3). All analyses were done
with the Statistica v5.5 program (StatSoft, Tulsa, OK,
USA).
RESULTS AND DISCUSSION
The proximate compositions of the V. unguiculata
L. Walp var. IT86D-719 WM, PM and PC were
generally similar to reported levels for cowpeas
(Table 1). Protein content in the WM was similar to
that found in other cowpea (V. unguiculata) varieties
(180–310 g kg−1 DM)7,17,18 but slightly higher than
levels previously reported for cowpeas cultivated in
Yucatan (225–241 g kg−1 DM)19 . The dehulling
process increased protein content in the PM, though
its protein value was not statistically different from that
of the WM. This agrees with Aremu,20 although his
results were not statistically evaluated. The increase
in protein content observed in the present study was
attributed to changes in proximate composition during
dehulling. Crude fiber content dropped by 11.8 g kg−1
Table 1. Proximate composition (g kg−1 dry matter)a of cowpea
(Vigna unguiculata L. Walp var. IT86D-719) whole meal (WM),
processed meal (PM) and protein concentrate (PC)
Item
WM
PM
PC
Moisture
91.90 ± 1.50a
77.27 ± 0.50b
77.48 ± 1.00b
Ash
32.39 ± 0.61a
25.84 ± 0.98b
32.27 ± 0.28a
Crude fat 18.07 ± 2.72b
11.45 ± 0.29c
58.47 ± 0.22a
Crude
247.53 ± 7.50b 257.00 ± 1.91b 786.00 ± 3.40a
protein
Crude
35.40 ± 2.90a
24.23 ± 1.03b
3.97 ± 0.49c
fibre
NFEb
666.61 ± 6.79a 681.48 ± 2.71a 119.29 ± 7.00b
a
Values are mean ± standard deviation of three determinations.
Different letters in the same row indicate significantly different
(P < 0.05) means.
b Nitrogen-free extract, estimated by difference.
114
DM, raising the net proportion of protein, despite the
seed coat containing 112.5 ± 2.4 g protein kg−1 DM
or 53.7 g protein kg−1 total whole grain protein.
The PC had significantly (P < 0.05) higher protein
(786 g kg−1 ) and fat (58.47 g kg−1 ) contents and
lower fibre and carbohydrate levels than the meals.
Protein concentrates of similar quality have been
obtained from other legumes using the same isoelectric
precipitation technique.6,21,22 Protein isolates (or
concentrates) from legumes normally have a high lipid
content, due to a binding mechanism between protein
and lipid that may result from emulsification of the
lipid by the protein.23
The WM, PM and PC amino acid profiles
(Table 2) show that the first limiting amino acids
in all three fractions were the sulphur amino acids
(methionine/cysteine). This was expected, since it
confirms the results of a number of other legume
protein quality studies.6,17,18,20,24 Tryptophan is the
second limiting amino acid in legume seeds. Its
levels in the WM, PM and PC do not meet the
requirements for children of 2–5 years but do meet
those for children of 10–12 years and adults of 18+
years.25 In comparison with other studies, the present
tryptophan levels (8.0 g kg−1 ) are similar to the average
for cowpeas reported by Kelly (cited in Ref. 26)
but lower than that reported by Maia et al.18 for six
Brazilian cowpea varieties (8.93 g kg−1 ). The levels
found here may have resulted from the processing
method used or simply from variations in genetics,
environmental factors and/or seed maturation.26
Worth noting is that lysine content dropped
significantly (P < 0.05) during the soaking and
dehulling process, from 68.0 ± 0.60 g kg−1 (WM) to
58.4 ± 0.75 g kg−1 (PM). However, there was little
change between lysine values in the PM and PC
(P > 0.05). Lysine values in all three fractions meet
the requirements for children of 2–12 years and adults
of 18+ years.25
Levels of the remaining essential amino acids in
the three fractions meet the requirements for all
children and adults. Although threonine and histidine
did decrease significantly (P < 0.05) in the PM and
PC, they remained within normal ranges for these
amino acids in legumes. This differs from the findings
of Fern´andez-Quintela et al.6 and Aremu,20 who
reported higher values for these amino acids in cowpea
protein concentrate. Leucine and isoleucine values
were similar (P > 0.05) in all three fractions, with
leucine/isoleucine ratios ranging from 1.74 to 2.18.
The lowest ratio was in the PC, making it appropriate
for complementing the cereal diets that predominate
among the rural people of Yucatan. This ratio was
similar to that (1.85) found by Chan and Phillips24
in the globulin-enriched fraction of V. unguiculata cv.
California Blackeye No 5.
Each fraction’s essential amino acid profile coincides
with those reported for cowpeas in the literature, and
any one of them could be used as a sole protein
source. Legume seeds, however, are usually eaten as
J Sci Food Agric 87:112–119 (2007)
DOI: 10.1002/jsfa
Bioactive factor content of cowpea raw meal and protein concentrate
Table 2. Amino acid content (g kg−1 protein)a of cowpea (Vigna unguiculata L. Walp var. IT86D-719) whole meal (WM), processed meal (PM) and
protein concentrate (PC) in comparison with daily requirements for children (2–5 and 10–12 years) and adults (18+ years)
Requirements25
Amino acid
Essential
Lysine
AAAb
SAAb
Threonine
Leucine
Isoleucine
Valine
Tryptophanc
Histidine
Non-essential
Arginine
Aspartic acid
Glutamic acid
Serine
Glycine
Alanine
Proline
WM
PM
PC
Children (2–5)
Children (10–12)
Adults (18+)
68.0 ± 0.60a
60.2 ± 0.53b
8.8 ± 0.08b
74.1 ± 0.52a
70.1 ± 0.62a
32.1 ± 0.28a
40.5 ± 0.36a
8.0 ± 0.01a
49.7 ± 0.44a
58.4 ± 0.75b
60.2 ± 0.44b
13.0 ± 0.11a
41.9 ± 0.79b
71.1 ± 0.72a
37.9 ± 0.62a
47.5 ± 0.76a
8.0 ± 0.01a
37.6 ± 0.98b
50.8 ± 0.67b
74.2 ± 1.21a
14.5 ± 0.25a
41.5 ± 0.55b
69.2 ± 0.94a
39.7 ± 0.56a
48.2 ± 0.58a
8.0 ± 0.00a
34.0 ± 0.39b
58
63
25
34
66
28
35
11
19
44
22
22
28
44
28
25
9
–
16
–
19
9
19
13
13
5
16
98.6 ± 0.71a
110.2 ± 0.89a
173.8 ± 1.26a
54.10 ± 0.50a
42.1 ± 0.37a
36.9 ± 0.33a
61.1 ± 0.55a
49.0 ± 0.71b
109.5 ± 1.92a
161.8 ± 1.92a
60.1 ± 0.61a
43.1 ± 0.95a
49.0 ± 0.77a
61.6 ± 1.15a
65.7 ± 0.54b
109.3 ± 0.69a
154.7 ± 2.34a
60.2 ± 0.55a
41.3 ± 0.44a
47.6 ± 0.47a
67.1 ± 0.53a
a
Values (except for tryptophan) are mean ± standard deviation of five determinations. Different letters in the same row indicate significantly different
(P < 0.05) means.
b AAA, total aromatic amino acids; SAA, total sulphur amino acids.
c
Values are mean ± standard deviation of three determinations.
Table 3. Trypsin inhibitor activitya in cowpea (Vigna unguiculata L. Walp var. IT86D-719) whole meal (WM), processed meal (PM) and protein
concentrate (PC)
Trypsin inhibitor activityb
TUI kg−1 dry matter (DM)
TUI kg−1 protein
g enzyme inhibited kg−1 DM
a
b
WM
PM (lyophilised)
PM (dry heat treated)
PC
(34.52 ± 0.06) × 106 a
(141.52 ± 0.24) × 106 a
49.08 ± 0.43a
(34.60 ± 0.06) × 106 a
(134.50 ± 0.23) × 106 ab
49.52 ± 0.15a
(33.27 ± 0.06) × 106 b
(134.00 ± 0.12) × 106 b
48.75 ± 0.59a
(27.86 ± 0.06) × 106 c
(35.45 ± 0.73) × 106 c
44.04 ± 0.84b
Values are mean ± standard deviation of three determinations. Different letters in the same row indicate significantly different (P < 0.05) means.
TUI, trypsin units inhibited. One TU is defined as 0.01 at A410 under the assay conditions (pH 8.1, 37 ◦ C, 4 mL assay volume, porcine trypsin).
a complement to cereal-rich diets, thus providing a
complete amino acid profile. If any one of the three
fractions was used as the sole protein source in human
or animal diets, it would need to be enriched with this
legume’s limiting amino acids.
Of the non-essential amino acids, aspartic acid and
glutamic acid were abundant in all three fractions.
This is common in cowpea meals and protein
concentrates17,18,24,27 and may adversely affect the
solubility of the protein concentrate.27 Overall, the
PC’s amino acid profile was similar to that of the
PM. The exception was the aromatic amino acids
(tyrosine + phenylalanine), which rose in the PC
because tyrosine increased (28.8 ± 0.40 g kg−1 ).
Trypsin inhibitor activity (Table 3) was statistically similar (P > 0.05) between the WM and the
lyophilised PM in all three quantifications (i.e. TUI
kg−1 DM, TUI kg−1 protein, g enzyme inhibited
kg−1 DM), though there were very minor differences
between the lyophilised and dry heat-treated PMs.
Trypsin inhibitor activity in the WM was comparable
to that reported by other researchers,12,28,29 though
J Sci Food Agric 87:112–119 (2007)
DOI: 10.1002/jsfa
Maia et al.18 found lower trypsin inhibitor activity
values (expressed as g kg−1 ) using Brazilian cowpea
cultivars. When compared with soya bean cultivars, the
present cowpea trypsin inhibitor levels were approximately five times lower than those of some soya bean
meals.12 Nevertheless, Armour et al.14 reported results
(between 39.5 and 49.7 TUI kg−1 DM) similar to
those for cowpeas in nine of the 26 soya bean cultivars
they studied. The differences between different soya
bean cultivars as well as between them and cowpeas
may be due to environmental differences in the areas
of cultivation.29
Cold soaking did not reduce trypsin inhibitor
activity in the PM (Table 3). This agrees with the
findings of Wang et al.30 and Ogun et al.,4 who
concluded that cold soaking was not an efficient
method because it did not eliminate trypsin inhibitor
activity. Wang et al.30 observed that cold soaking for
18 h did not effectively remove trypsin inhibitor from a
soya bean cultivar. Ogun et al.4 stated that soaking at
22 ◦ C for 12 h did not affect trypsin inhibitor activity
when compared with a control treatment; however,
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L Olivera-Castillo et al.
this was a combined result for four cultivars, with no
analysis of variation by cultivar.
Although cold soaking may not eliminate significant
amounts of trypsin inhibitor activity, the present
results indicate that dry heat treatment at 40 ◦ C also
did not substantially lower trypsin inhibitor activity.
Indeed, the differences between the lyophilised and
dry heat-treated PMs was not significant in two of the
quantifications (TUI kg−1 protein, g enzyme inhibited
kg−1 DM) and barely significant in the other (TUI
kg−1 DM) (Table 3). Apparently, g enzyme inhibited
kg−1 DM most accurately reflects trypsin inhibitor
activity and may be the most appropriate way of
quantifying this antinutritional factor.
In all three quantifications the humid heat (80 ◦ C) of
the concentration process significantly reduced trypsin
inhibitor activity in the PC compared with levels in
the WM and PM. It did not, however, completely
eliminate trypsin inhibitor activity as was reported by
Armour et al.14 for some Japanese soya bean (Glycine
max) cultivars, meaning that the trypsin inhibitor in
V. unguiculata L. Walp (ITD86-719) is partially heatresistant.
α-Amylase inhibitor was detected in very low
concentrations in the WM, PM and PC (Table 4).
When the values were expressed as g equivalent kg−1
DM, they were statistically different (P < 0.05), with
the highest inhibitor activity in the PM and the lowest
in the PC. Piergiovanni13 also reported higher activity
in the PM when dehulling light-coloured cowpea
seeds, explaining that this may result from inhibition
of the α-amylase inhibitor by polyphenolic compounds
in the testa. That is why inhibitor activity was higher
in whole seeds and why there was less inhibitor activity
in seeds with darker testae. Grant et al.31 reported a
value of 0.2 ± 0.1 g equivalent kg−1 DM for whole
grain cowpeas, using the same method as here, and
considered this inhibitor activity to be low compared
with that of kidney bean (Phaseolus vulgaris), haricot
bean (P. vulgaris), pinto bean (P. vulgaris) and runner
bean (Phaseolus coccineus).
When expressed as g equivalent kg−1 protein,
α-amylase inhibitor activity was again significantly
different (P < 0.05) among the three fractions, with
the highest value in the PM and the lowest in the PC.
This means that V. unguiculata L. Walp var. IT86D719 has a very low concentration of this protease
inhibitor, particularly in the PC.
When estimated as haemagglutination versus hamster blood cells, lectin activity was detected in the WM
and PM but not in the PC (Table 4). No lectin activity
was detected in any of the fractions when bovine or
human type A+ red blood cells were used. For the WM
this result is comparable to that reported by Armour
et al.14 for four Japanese soya bean cultivars that
had the lowest lectin activity values (2.4 ± 0.8 kg−1
DM) of 30 cultivars studied, with values ranging from
9.6 ± 3.2 to 4.8 ± 1.6 kg−1 DM. They concluded that
soya bean lectin activity is relatively heat-resistant
and consequently that heat treatment reduces but
does not eliminate it. Other tropical grains and kidney
bean31,32 have reported lectin activity levels lower than
in the cowpeas studied here; in fact, lectin activity in
kidney bean was completely eliminated after heating
for 5 min at 100 ◦ C. However, Maia et al.18 reported
higher lectin activity levels than in the present study
in six cultivars of Brazilian cowpeas using trypsinated
blood cell treatment. These results are only generally
comparable with the present ones, because the authors
used rabbit blood cells (trypsinated rat, bovine, human
and hamster blood was used in the present study) and
no heat treatment was applied, which may affect lectin
affinity for carbohydrates in the blood cell membrane.
As mentioned above, no appreciable lectin activity
was observed in the PC, making it significantly
(P < 0.05) lower than in the WM. This is most likely
a result of the soaking process. Fern´andez-Quintela
et al.6 reported similar results in that they observed
no detectable lectin activity in protein concentrates
Table 4. Antinutritional factorsa (except trypsin inhibitors) in cowpea (Vigna unguiculata L. Walp var. IT86D-719) whole meal (WM), processed meal
(PM) and protein concentrate (PC)
Antinutritional factor
α-Amylase inhibitor
g equivalent kg−1 DM
g equivalent kg−1 protein
Lectin activity
HU kg−1 DM
g lectin equivalent kg−1 DM
Tannins
g tannic acid kg−1 DM
g tannic acid kg−1 protein
Phytates
g phytic acid kg−1 DM
g phytic acid kg−1 protein
Cyanogenic glucosides
g HCN kg−1 DM
WM
PM
PC
(8.4 ± 0.4) × 10−3 b
(3.4 ± 0.84) × 10−2 b
(1 ± 0.08) × 10−2 a
(4.8 ± 0.84) × 10−2 a
(6 ± 0.2) × 10−3 c
(8.1 ± 0.7) × 10−3 c
0.24 ± 0.04a
2.5 ± 0.41a
(1 ± 0.08) × 10−5 b
(6 ± 0.30) × 10−3 b
ND
ND
1.94 ± 0.04a
7.93 ± 0.16a
1.75 ± 0.02b
6.82 ± 0.06b
1.12 ± 0.02c
1.42 ± 0.02c
6.86 ± 0.01a
28.1 ± 0.09a
5.11 ± 0.02c
19.8 ± 0.53b
6.24 ± 0.02b
7.93 ± 0.020c
ND
ND
ND
a
Values are mean ± standard deviation of three determinations. Different letters in the same row indicate significantly different (P < 0.05) means.
DM, dry matter; HU, haemagglutinating activity unit; ND, not detected.
116
J Sci Food Agric 87:112–119 (2007)
DOI: 10.1002/jsfa
Bioactive factor content of cowpea raw meal and protein concentrate
from peas (Pisum sativum), faba bean (Vicia faba) and
soya bean, which they explained as being caused by
concentrate preparation; they used the same isoelectric
point method as in the present study.
Tannin was present in the WM, PM and PC fractions (Table 4). Tannin levels dropped progressively
as the meal was processed, producing significant differences among fractions whether expressed as g tannic
acid kg−1 DM or kg−1 protein. Ogun et al.4 reported
similar tannin content results for one raw cultivar
(Kano 1696) of the four cultivars they studied. After
processing (dehulling, cold soaking, hot soaking), no
tannins were detected in any of the four cultivars. Cold
soaking caused a non-significant decrease in tannins
in comparison with the raw grains, and hot soaking
led to a greater but still non-significant drop in tannins
in comparison with the raw grains. The tannin levels
in the present PM may have originated in tannins
from other parts of the grain besides the testa or from
residual hulls. If the latter is true, dehulling efficiency
was notably lower in the present study than in that of
Ogun et al.4
Despite slight variations in different studies,
dehulling and the protein concentration process clearly
affect tannin content in the final product. For example,
Fern´andez-Quintela et al.6 observed that antinutritional factor content, including tannins, drops after
protein concentrate preparation. Because the highest tannin concentrations in V. unguiculata and other
seeds are generally found in the testa,4,33 the lack of
detectable tannins in the PC probably resulted from
removal of the testa prior to protein extraction, as well
as retention of low-molecular-weight polyphenols34 in
the water-soluble (pH 4.3) phase.
There were significant differences (P < 0.05) in
phytate content among the WM, PM and PC
fractions on both a DM and protein basis (Table 4),
though each basis exhibited a different pattern.
The phytate concentration on a DM basis in the
PM was significantly lower (P < 0.05) than in the
WM and PC. When compared in terms of protein,
however, the concentration decreased progressively,
with significantly lower (P < 0.05) values between the
WM and PM and between the PM and PC. The
phytate concentration value (DM and protein basis)
in the WM was comparable to those found in many
other legumes.33,35,36 Hidvegi and Lasztity36 reported
that phytic acid content in cowpea meal can vary from
2.9 to 8.6 g kg−1 , while in soya protein concentrate
they reported a phytic acid content of 8.2 g kg−1 .
Oluwatosin29 found higher dry matter basis phytic
acid values in cowpeas than in the present study,
with values ranging from 25.07 to 16.05 g kg−1 . This
discrepancy may be explained by the fact that some
of their varieties, including var. IT86D-534 from the
same source as var. IT86D-719, resulted from recent
breeding programmes aimed at improving resistance
to pests and diseases.
Processing (i.e. dehulling and soaking) notably
affected phytic acid concentration in the present
J Sci Food Agric 87:112–119 (2007)
DOI: 10.1002/jsfa
study. Ogun et al.4 , however, reported that it had no
significant effect on phytic acid, with average values of
1.2 ± 0.02 g kg−1 with dehulling and 1.1 ± 0.02 g kg−1
with cold soaking for four cultivars; these are quite
different from the present results for the PM. This
contrast may be due to the extraction method or simply
to phytate solubility in var. IT86D-719. Comparison
of phytate concentration variation in terms of protein
content is not possible, since Ogun et al.4 did not
express their data on that basis.
On a protein basis the present PC had 3.5 times
less phytic acid than the WM and 2.5 times less
than the PM, with its value of 7.93 g kg−1 being
relatively close to the values of 9.03 g kg−1 for soya
bean isolate and 10.1 g kg−1 for protein concentrate
reported by Hidvegi and Lasztity.36 The difference
between the meals and the PC is probably due
to most of the phytates being in the cotyledon,
meaning that they would have been extracted at pH
9. However, the remaining phytates may be highly
water-soluble and therefore did not precipitate during
protein concentrate preparation. Phytic acid was not
completely eliminated in the PC, meaning that the
processing method did not remove it all and/or the raw
material had high concentrations. This results from the
phytic acid concentration in protein concentrates or
isolates being dependent on the raw materials from
which they are prepared as well as the processing
method. The final concentration may also be affected
by the protein–phytic acid interaction, which is highly
pH-dependent. A pH above a protein’s isoelectric
point can diminish the protein–phytic acid interaction,
while a pH below this point can raise the interaction,
though binding begins to decrease below pH 2.5.
No cyanogenic glucosides were detected in the
WM and thus they were absent in the PM and PC
fractions. This contrasts with the result of Colom´e
et al.,19 who found 2.9 mg HCN kg−1 in whole V.
unguiculata meal. The difference between the present
data and those of Colom´e et al.19 is probably due
to the fact that cyanogenic glucoside levels can vary
considerably depending on a crop’s location and
environmental conditions, as is the case with many
other antinutritional factor contents.
CONCLUSIONS
Overall, V. unguiculata L. Walp (IT86D-719) meal
appeared to contain only moderate or low levels
of potentially bioactive or antinutritional factors,
indicating that meals (WM and PM) from such
cowpeas grown in Yucatan, Mexico, or a protein
concentrate (PC) derived from them, are potentially
good foodstuffs. Given that neither the meals nor the
PC have high levels of adverse bioactive/antinutritional
factors, they can be effectively used as sources of
dietary protein, energy or essential amino acids with
only minimal processing. However, they cannot be
consumed as a sole protein source because they are
limited in sulphur amino acids and therefore must be
117
L Olivera-Castillo et al.
combined with cereals to balance their amino acid
profile.
Trypsin inhibitors, α-amylase inhibitor and lectins
in many legume seeds are albumin-like proteins
and thus soluble in aqueous solution. The low
concentrations of some antinutrients and the absence
of lectin observed in the cowpea PC (pH 4.3 insoluble)
would be consistent with the constituent bioactive
factors being albumin-like proteins. The persistence of
some trypsin inhibitor in the PC may be a carry-over
from its high concentration in the WM. Repeating
the solubilisation/precipitation procedures used here
with the PC may further reduce trypsin inhibitor
levels. Completely eliminating all antinutrients in
the PC, however, may not necessarily be the most
advantageous overall approach, since at certain levels
they can aid in disease prevention. In vivo nutritional
studies will be required to firmly establish these
properties and better understand this legume’s high
potential.
ACKNOWLEDGEMENTS
The authors thank C´esar Puerto-Castillo and Wilberth
´ for their laboratory assistance, Laura
Ch´e-Leon
Escobar-Brillones for help with the statistical analysis,
and Dr Luis Chel-Guerrero for facilitating the
tryptophan analysis. This research was done as
part of a British Council/Mexico HEL Program
(MXC/991/83). Support was also provided by the
Scottish Executive Environment and Rural Affairs
Department and the International Foundation for
Science (grant E/3171-1).
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119
J Sci Food Agric 87:112–119 (2007)
Composition and bioactive factor
content of cowpea (Vigna unguiculata L.
Walp) raw meal and protein concentrate
Leticia Olivera-Castillo,1∗ Fabiola Pereira-Pacheco,2 Erik Polanco-Lugo,2
Miguel Olvera-Novoa,1 Jose´ Rivas-Burgos3 and George Grant4
1 Centro
´ y de Estudios Avanzados del IPN, Unidad Merida, Antigua Carretera a Progreso Km 6, 97310 Merida, Yucatan,
de Investigacion
Mexico
2 Facultad de Ingenieria Qu´ımica, Universidad Autonoma
´
´
de Yucatan, Av. Juarez
No 421, Cd. Industrial, 97288 Merida, Yucatan, Mexico
3 Instituto Tecnologico de Merida, Antigua Carretera Progreso Km 5, 97118 Merida, Yucatan, Mexico
4 The Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK
Abstract: Analysis was done of the composition and bioactive factor content of whole meal, processed meal and
protein concentrate from a cowpea cultivar (Vigna unguiculata L. Walp var. IT86D-719) grown in Yucatan, Mexico
and of changes in these parameters after soaking and dehulling. Both meals had a high protein content (247.53 and
257 g kg−1 dry matter (DM) respectively). The protein concentrate was rich in protein (786 g kg−1 DM) and lipids
(58.47 g kg−1 DM) and had an amino acid profile similar to that of the processed meal. The amino acid profiles
of the meals almost covered human dietary requirements based on FAO/WHO/UNU-suggested profiles but were
deficient in sulphur amino acids. Trypsin inhibitor activity was high in both meals compared with levels found in
previous studies. Trypsin inhibitor activity in the concentrate was not eliminated but was significantly reduced.
Lectin activity, tannin levels, phytate levels and α-amylase inhibitor activity were relatively low in the meals, and
cyanogenic glucosides were not detected. Residual amounts of α-amylase inhibitors, tannins and phytate were
observed in the concentrate, and lectin activity was not detected. Results indicate that V. unguiculata L. Walp var.
IT86D-719 meals and protein concentrate are good potential foodstuffs in the Yucatan region.
2006 Society of Chemical Industry
Keywords: Vigna unguiculata L. Walp; proximate analysis; amino acid composition; antinutritional factors;
Yucatan, Mexico
INTRODUCTION
Legume grains are a primary source of dietary
protein in many regions of the world.1 However,
yields vary greatly according to plant species,
environmental conditions and horticultural practices.
The actual nutritional value of these crops is also
compromised by an apparently low digestibility of
their constituent proteins and by adverse effects of
seed components on body metabolism, particularly
bioactive/antinutritional factors such as lectins, trypsin
inhibitors, tannins, etc.1,2
Most legume seeds therefore require extensive
processing before they can be used safely and
effectively. Various methods have been developed
to improve the nutritional properties of legume
seed products, including sprouting,3 soaking and
cooking,4 extrusion5 and isolation of the major storage
(globulin-like) proteins, i.e. protein concentration.
Protein concentrate quality may vary, however,
depending on the exact preparation conditions,
and thus new legume protein concentrates require
chemical characterisation of their composition and
bioactive/antinutritional factor content. Despite this
variation, protein concentrates generally have good
nutritional quality, partially because globulin-like
proteins have low antinutritional factor levels.6
Cowpeas (Vigna unguiculata) grow well in a diverse
range of conditions and environments and contain only
moderate levels of bioactive/antinutritional factors.7
The National Institute of Forestry, Agricultural
and Livestock Research (Instituto Nacional de
Investigaciones Forestales, Agr´ıcolas y Pecuarias
(INIFAP)) located in Uxmal, Yucatan, Mexico
recently identified V. unguiculata L. Walp var. IT86D719, a variety created at the International Institute
for Tropical Agriculture (IITA) in Ibadan, Nigeria, as
having good agronomic potential for use as a crop
plant in the Yucatan Peninsula region. Indeed, it
was found to have a high protein and starch content
and a yield of up to 3.5 T ha−1 , significantly higher
∗
´ y de Estudios Avanzados del IPN, Unidad Merida, Antigua Carretera a Progreso Km 6,
Correspondence to: Leticia Olivera-Castillo, Centro de Investigacion
97310 Merida, Yucatan, Mexico
E-mail: olivera@mda.cinvestav.mx
Contract/grant sponsor: British Council/Mexico HEL Program; contract/grant number: MXC/991/83
Contract/grant sponsor: Scottish Executive Environment and Rural Affairs Department
Contract/grant sponsor: International Foundation for Science; contract/grant number: E/3171-1
(Received 25 November 2004; revised version received 7 October 2005; accepted 15 June 2006)
Published online 16 October 2006; DOI: 10.1002/jsfa.2684
2006 Society of Chemical Industry. J Sci Food Agric 0022–5142/2006/$30.00
Bioactive factor content of cowpea raw meal and protein concentrate
than that achieved with other legumes grown in the
region. Despite its promise, usage of cowpea by the
regional population remains low; unfamiliarity with
this legume, its horticulture and possible uses means
that there is only a limited local market for it.
As part of a developmental programme, V.
unguiculata L. Walp var. IT86D-719 seeds were
provided to small-scale farmers in rural Yucatan
and grown in accordance with local horticultural
practices. The composition, amino acid profile and
bioactive/antinutritional factor content of whole meal,
processed meal and protein concentrate prepared from
this legume are reported in this study. Additional
information is provided on changes in the proximal
and antinutritional composition resulting from the
protein concentration process.
EXPERIMENTAL
Seed preparation
Vigna unguiculata L. Walp (IT86D-719) seeds
from INIFAP were cultivated in the rural area of
Santa Elena, Yucatan, harvested when mature and
dry, cleaned and stored at 4 ◦ C until processing.
Representative samples of the seeds were ground in
a hammer mill fitted with a 1 mm2 mesh. Whole V.
unguiculata seeds (3 kg) were soaked (1:4 w/v) for 16 h
at 25 ◦ C in sodium bisulphite (2 g L−1 ) solution. The
seeds were then cracked in a manual mill and the
hulls separated off by water flotation (dehulling). The
cotyledons were cleaned and then dried in a lyophiliser,
though one sample of fresh cotyledons was dried in
a convection oven at 40 ◦ C to determine if dry heat
reduced trypsin inhibitor levels.
Protein concentrate preparation
Protein extraction was done using a modified version
of the isoelectric method.8 Briefly, the cotyledons were
passed through an industrial mill (Koch mod. C352Z,
Kansas City, MO, USA) to produce a homogenous
dough, which was then ground (1:3 w/v) in water in
a colloidal mill (Micron Mod. WB-1, M´exico, DF,
Mexico). The pH of the resulting slurry was adjusted
to 9 using 0.05 mol L−1 NaOH. After approximately
20 min the suspension was filtered through 0.150
and 0.106 mm2 meshes to remove fibrous material.
The remaining slurry was stored without agitation
at 4 ◦ C for 12 h to allow the starch to settle. The
supernatant was decanted and its pH adjusted to 4.33
with 0.5 mol L−1 HCl. It was then heated at 80 ◦ C for
10 min and stored for 12 h at 4 ◦ C. The precipitated
globulin-enriched fraction (i.e. protein concentrate)
was recovered by centrifugation (1500 × g, 12 min)
and subsequent drying in a convection oven at 40 ◦ C
for 24 h.
Proximate composition
Moisture, ash, crude fibre and crude fat contents
in the whole meal, processed meal and protein
concentrate were determined according to standard
J Sci Food Agric 87:112–119 (2007)
DOI: 10.1002/jsfa
methods.9 Nitrogen content was estimated by gas
chromatography using a ThermoQuest NCS analyser
(ThermoQuest Flash EA 1112, Rodano, Italy). Crude
protein content was calculated as N × 6.25. All
analyses were run in triplicate.
Amino acid analysis
Amino acid analysis of the defatted meals and
defatted protein concentrate was done using a
four-step Pico-Tag method.10 In the Pico-Tag
station, hydrolysis was done using 3 mol L−1
HCl under vacuum at 104 ◦ C for 24 h, followed by redrying with ethanol/water/triethylamine
(2:2:1 v/v/v) solution and derivatisation with
ethanol/triethylamine/water/phenylisothiocyanate
(7:1:1:1 v/v/v/v) reagent. Once derivitised, aliquots
were subjected to reverse phase liquid chromatography
in a Waters high-performance liquid chromatography
(HPLC) system (Waters Corporation, Milford, MA,
USA). Tryptophan was determined by a colorimetric
method.11 All analyses were run in triplicate.
Analysis of antinutritional factors in meals and
protein concentrate
Trypsin inhibitor activity, α-amylase inhibitor content,
lectin activity, tannic acid levels, phytate content and
cyanogenic glucoside levels were determined in the
whole meal (WM), processed meal (PM) and protein
concentrate (PC) as follows.
Trypsin inhibitor activity was assayed by the method
of Liu and Markarkis12 using benzoyl-DL-arginine-pnitroanilide (BAPNA) as substrate and porcine trypsin
(Type II-S, Sigma Chemical St. Louis, Mo, USA).
One trypsin unit (TU) was defined as 0.01 at A410
under the assay conditions, and trypsin inhibitor
activity was expressed as trypsin units inhibited (TUI)
kg−1 dry matter (DM) and kg−1 protein. Trypsin
inhibitor activity in the WM, PM and PC was also
determined from diluted samples of the fractions with
40–60% inhibition of enzyme activity and expressed
as g enzyme inhibited kg−1 DM.
α-Amylase inhibitor content was determined by
the procedure of Piergiovanni13 and expressed as g
amylase inhibited kg−1 DM and kg−1 protein.
Lectin activity (haemagglutination) was assayed by
the serial dilution method of Armour et al.14 using
human, bovine and hamster red blood cells previously
treated with trypsin. One haemagglutinating activity
unit (HU) was defined as that contained in the amount
of sample in the final dilution which caused 50%
agglutination of the blood cells. Lectin activity was
also determined using pure Phaseolus vulgaris lectin
(Sigma Chemical) as a standard and expressed as g
lectin equivalent kg−1 DM.
Tannic acid levels were determined using a
spectrophotometric method15 and expressed as g
tannic acid kg−1 DM and kg−1 protein.
Phytate content was determined by an anion
exchange method.9 Phytate was extracted from
triplicate dry samples using diluted HCl (24 g L−1 ).
113
L Olivera-Castillo et al.
The extract was then mixed with ethylene diamine
tetraacetic acid (EDTA)/NaOH solution and placed
in an ion exchange column. The phytate was eluted
with 0.7 mol L−1 NaCl solution and wet digested
with HNO3 /H2 SO4 mixture to release phosphorus,
which was measured calorimetrically at 640 nm and
expressed as g phytic acid kg−1 DM and kg−1 protein.
Cyanogenic glucoside levels were determined using
the method of Lucas and Sotelo,16 based on the
specific Guinard reaction, and expressed as g HCN
kg−1 DM.
Statistical analysis
With the exception of lectin activity, results were
subjected to one-way analysis of variance (ANOVA) at
P < 0.05. Differences between means were evaluated
by the Newman–Keuls test; normality was tested
using the Wilk–Shapiro test. When necessary, values
were normalised using natural logarithms. Lectin
activity results were compared using the Student t
test (P < 0.05). Means for all data were calculated
from three replicates (n = 3). All analyses were done
with the Statistica v5.5 program (StatSoft, Tulsa, OK,
USA).
RESULTS AND DISCUSSION
The proximate compositions of the V. unguiculata
L. Walp var. IT86D-719 WM, PM and PC were
generally similar to reported levels for cowpeas
(Table 1). Protein content in the WM was similar to
that found in other cowpea (V. unguiculata) varieties
(180–310 g kg−1 DM)7,17,18 but slightly higher than
levels previously reported for cowpeas cultivated in
Yucatan (225–241 g kg−1 DM)19 . The dehulling
process increased protein content in the PM, though
its protein value was not statistically different from that
of the WM. This agrees with Aremu,20 although his
results were not statistically evaluated. The increase
in protein content observed in the present study was
attributed to changes in proximate composition during
dehulling. Crude fiber content dropped by 11.8 g kg−1
Table 1. Proximate composition (g kg−1 dry matter)a of cowpea
(Vigna unguiculata L. Walp var. IT86D-719) whole meal (WM),
processed meal (PM) and protein concentrate (PC)
Item
WM
PM
PC
Moisture
91.90 ± 1.50a
77.27 ± 0.50b
77.48 ± 1.00b
Ash
32.39 ± 0.61a
25.84 ± 0.98b
32.27 ± 0.28a
Crude fat 18.07 ± 2.72b
11.45 ± 0.29c
58.47 ± 0.22a
Crude
247.53 ± 7.50b 257.00 ± 1.91b 786.00 ± 3.40a
protein
Crude
35.40 ± 2.90a
24.23 ± 1.03b
3.97 ± 0.49c
fibre
NFEb
666.61 ± 6.79a 681.48 ± 2.71a 119.29 ± 7.00b
a
Values are mean ± standard deviation of three determinations.
Different letters in the same row indicate significantly different
(P < 0.05) means.
b Nitrogen-free extract, estimated by difference.
114
DM, raising the net proportion of protein, despite the
seed coat containing 112.5 ± 2.4 g protein kg−1 DM
or 53.7 g protein kg−1 total whole grain protein.
The PC had significantly (P < 0.05) higher protein
(786 g kg−1 ) and fat (58.47 g kg−1 ) contents and
lower fibre and carbohydrate levels than the meals.
Protein concentrates of similar quality have been
obtained from other legumes using the same isoelectric
precipitation technique.6,21,22 Protein isolates (or
concentrates) from legumes normally have a high lipid
content, due to a binding mechanism between protein
and lipid that may result from emulsification of the
lipid by the protein.23
The WM, PM and PC amino acid profiles
(Table 2) show that the first limiting amino acids
in all three fractions were the sulphur amino acids
(methionine/cysteine). This was expected, since it
confirms the results of a number of other legume
protein quality studies.6,17,18,20,24 Tryptophan is the
second limiting amino acid in legume seeds. Its
levels in the WM, PM and PC do not meet the
requirements for children of 2–5 years but do meet
those for children of 10–12 years and adults of 18+
years.25 In comparison with other studies, the present
tryptophan levels (8.0 g kg−1 ) are similar to the average
for cowpeas reported by Kelly (cited in Ref. 26)
but lower than that reported by Maia et al.18 for six
Brazilian cowpea varieties (8.93 g kg−1 ). The levels
found here may have resulted from the processing
method used or simply from variations in genetics,
environmental factors and/or seed maturation.26
Worth noting is that lysine content dropped
significantly (P < 0.05) during the soaking and
dehulling process, from 68.0 ± 0.60 g kg−1 (WM) to
58.4 ± 0.75 g kg−1 (PM). However, there was little
change between lysine values in the PM and PC
(P > 0.05). Lysine values in all three fractions meet
the requirements for children of 2–12 years and adults
of 18+ years.25
Levels of the remaining essential amino acids in
the three fractions meet the requirements for all
children and adults. Although threonine and histidine
did decrease significantly (P < 0.05) in the PM and
PC, they remained within normal ranges for these
amino acids in legumes. This differs from the findings
of Fern´andez-Quintela et al.6 and Aremu,20 who
reported higher values for these amino acids in cowpea
protein concentrate. Leucine and isoleucine values
were similar (P > 0.05) in all three fractions, with
leucine/isoleucine ratios ranging from 1.74 to 2.18.
The lowest ratio was in the PC, making it appropriate
for complementing the cereal diets that predominate
among the rural people of Yucatan. This ratio was
similar to that (1.85) found by Chan and Phillips24
in the globulin-enriched fraction of V. unguiculata cv.
California Blackeye No 5.
Each fraction’s essential amino acid profile coincides
with those reported for cowpeas in the literature, and
any one of them could be used as a sole protein
source. Legume seeds, however, are usually eaten as
J Sci Food Agric 87:112–119 (2007)
DOI: 10.1002/jsfa
Bioactive factor content of cowpea raw meal and protein concentrate
Table 2. Amino acid content (g kg−1 protein)a of cowpea (Vigna unguiculata L. Walp var. IT86D-719) whole meal (WM), processed meal (PM) and
protein concentrate (PC) in comparison with daily requirements for children (2–5 and 10–12 years) and adults (18+ years)
Requirements25
Amino acid
Essential
Lysine
AAAb
SAAb
Threonine
Leucine
Isoleucine
Valine
Tryptophanc
Histidine
Non-essential
Arginine
Aspartic acid
Glutamic acid
Serine
Glycine
Alanine
Proline
WM
PM
PC
Children (2–5)
Children (10–12)
Adults (18+)
68.0 ± 0.60a
60.2 ± 0.53b
8.8 ± 0.08b
74.1 ± 0.52a
70.1 ± 0.62a
32.1 ± 0.28a
40.5 ± 0.36a
8.0 ± 0.01a
49.7 ± 0.44a
58.4 ± 0.75b
60.2 ± 0.44b
13.0 ± 0.11a
41.9 ± 0.79b
71.1 ± 0.72a
37.9 ± 0.62a
47.5 ± 0.76a
8.0 ± 0.01a
37.6 ± 0.98b
50.8 ± 0.67b
74.2 ± 1.21a
14.5 ± 0.25a
41.5 ± 0.55b
69.2 ± 0.94a
39.7 ± 0.56a
48.2 ± 0.58a
8.0 ± 0.00a
34.0 ± 0.39b
58
63
25
34
66
28
35
11
19
44
22
22
28
44
28
25
9
–
16
–
19
9
19
13
13
5
16
98.6 ± 0.71a
110.2 ± 0.89a
173.8 ± 1.26a
54.10 ± 0.50a
42.1 ± 0.37a
36.9 ± 0.33a
61.1 ± 0.55a
49.0 ± 0.71b
109.5 ± 1.92a
161.8 ± 1.92a
60.1 ± 0.61a
43.1 ± 0.95a
49.0 ± 0.77a
61.6 ± 1.15a
65.7 ± 0.54b
109.3 ± 0.69a
154.7 ± 2.34a
60.2 ± 0.55a
41.3 ± 0.44a
47.6 ± 0.47a
67.1 ± 0.53a
a
Values (except for tryptophan) are mean ± standard deviation of five determinations. Different letters in the same row indicate significantly different
(P < 0.05) means.
b AAA, total aromatic amino acids; SAA, total sulphur amino acids.
c
Values are mean ± standard deviation of three determinations.
Table 3. Trypsin inhibitor activitya in cowpea (Vigna unguiculata L. Walp var. IT86D-719) whole meal (WM), processed meal (PM) and protein
concentrate (PC)
Trypsin inhibitor activityb
TUI kg−1 dry matter (DM)
TUI kg−1 protein
g enzyme inhibited kg−1 DM
a
b
WM
PM (lyophilised)
PM (dry heat treated)
PC
(34.52 ± 0.06) × 106 a
(141.52 ± 0.24) × 106 a
49.08 ± 0.43a
(34.60 ± 0.06) × 106 a
(134.50 ± 0.23) × 106 ab
49.52 ± 0.15a
(33.27 ± 0.06) × 106 b
(134.00 ± 0.12) × 106 b
48.75 ± 0.59a
(27.86 ± 0.06) × 106 c
(35.45 ± 0.73) × 106 c
44.04 ± 0.84b
Values are mean ± standard deviation of three determinations. Different letters in the same row indicate significantly different (P < 0.05) means.
TUI, trypsin units inhibited. One TU is defined as 0.01 at A410 under the assay conditions (pH 8.1, 37 ◦ C, 4 mL assay volume, porcine trypsin).
a complement to cereal-rich diets, thus providing a
complete amino acid profile. If any one of the three
fractions was used as the sole protein source in human
or animal diets, it would need to be enriched with this
legume’s limiting amino acids.
Of the non-essential amino acids, aspartic acid and
glutamic acid were abundant in all three fractions.
This is common in cowpea meals and protein
concentrates17,18,24,27 and may adversely affect the
solubility of the protein concentrate.27 Overall, the
PC’s amino acid profile was similar to that of the
PM. The exception was the aromatic amino acids
(tyrosine + phenylalanine), which rose in the PC
because tyrosine increased (28.8 ± 0.40 g kg−1 ).
Trypsin inhibitor activity (Table 3) was statistically similar (P > 0.05) between the WM and the
lyophilised PM in all three quantifications (i.e. TUI
kg−1 DM, TUI kg−1 protein, g enzyme inhibited
kg−1 DM), though there were very minor differences
between the lyophilised and dry heat-treated PMs.
Trypsin inhibitor activity in the WM was comparable
to that reported by other researchers,12,28,29 though
J Sci Food Agric 87:112–119 (2007)
DOI: 10.1002/jsfa
Maia et al.18 found lower trypsin inhibitor activity
values (expressed as g kg−1 ) using Brazilian cowpea
cultivars. When compared with soya bean cultivars, the
present cowpea trypsin inhibitor levels were approximately five times lower than those of some soya bean
meals.12 Nevertheless, Armour et al.14 reported results
(between 39.5 and 49.7 TUI kg−1 DM) similar to
those for cowpeas in nine of the 26 soya bean cultivars
they studied. The differences between different soya
bean cultivars as well as between them and cowpeas
may be due to environmental differences in the areas
of cultivation.29
Cold soaking did not reduce trypsin inhibitor
activity in the PM (Table 3). This agrees with the
findings of Wang et al.30 and Ogun et al.,4 who
concluded that cold soaking was not an efficient
method because it did not eliminate trypsin inhibitor
activity. Wang et al.30 observed that cold soaking for
18 h did not effectively remove trypsin inhibitor from a
soya bean cultivar. Ogun et al.4 stated that soaking at
22 ◦ C for 12 h did not affect trypsin inhibitor activity
when compared with a control treatment; however,
115
L Olivera-Castillo et al.
this was a combined result for four cultivars, with no
analysis of variation by cultivar.
Although cold soaking may not eliminate significant
amounts of trypsin inhibitor activity, the present
results indicate that dry heat treatment at 40 ◦ C also
did not substantially lower trypsin inhibitor activity.
Indeed, the differences between the lyophilised and
dry heat-treated PMs was not significant in two of the
quantifications (TUI kg−1 protein, g enzyme inhibited
kg−1 DM) and barely significant in the other (TUI
kg−1 DM) (Table 3). Apparently, g enzyme inhibited
kg−1 DM most accurately reflects trypsin inhibitor
activity and may be the most appropriate way of
quantifying this antinutritional factor.
In all three quantifications the humid heat (80 ◦ C) of
the concentration process significantly reduced trypsin
inhibitor activity in the PC compared with levels in
the WM and PM. It did not, however, completely
eliminate trypsin inhibitor activity as was reported by
Armour et al.14 for some Japanese soya bean (Glycine
max) cultivars, meaning that the trypsin inhibitor in
V. unguiculata L. Walp (ITD86-719) is partially heatresistant.
α-Amylase inhibitor was detected in very low
concentrations in the WM, PM and PC (Table 4).
When the values were expressed as g equivalent kg−1
DM, they were statistically different (P < 0.05), with
the highest inhibitor activity in the PM and the lowest
in the PC. Piergiovanni13 also reported higher activity
in the PM when dehulling light-coloured cowpea
seeds, explaining that this may result from inhibition
of the α-amylase inhibitor by polyphenolic compounds
in the testa. That is why inhibitor activity was higher
in whole seeds and why there was less inhibitor activity
in seeds with darker testae. Grant et al.31 reported a
value of 0.2 ± 0.1 g equivalent kg−1 DM for whole
grain cowpeas, using the same method as here, and
considered this inhibitor activity to be low compared
with that of kidney bean (Phaseolus vulgaris), haricot
bean (P. vulgaris), pinto bean (P. vulgaris) and runner
bean (Phaseolus coccineus).
When expressed as g equivalent kg−1 protein,
α-amylase inhibitor activity was again significantly
different (P < 0.05) among the three fractions, with
the highest value in the PM and the lowest in the PC.
This means that V. unguiculata L. Walp var. IT86D719 has a very low concentration of this protease
inhibitor, particularly in the PC.
When estimated as haemagglutination versus hamster blood cells, lectin activity was detected in the WM
and PM but not in the PC (Table 4). No lectin activity
was detected in any of the fractions when bovine or
human type A+ red blood cells were used. For the WM
this result is comparable to that reported by Armour
et al.14 for four Japanese soya bean cultivars that
had the lowest lectin activity values (2.4 ± 0.8 kg−1
DM) of 30 cultivars studied, with values ranging from
9.6 ± 3.2 to 4.8 ± 1.6 kg−1 DM. They concluded that
soya bean lectin activity is relatively heat-resistant
and consequently that heat treatment reduces but
does not eliminate it. Other tropical grains and kidney
bean31,32 have reported lectin activity levels lower than
in the cowpeas studied here; in fact, lectin activity in
kidney bean was completely eliminated after heating
for 5 min at 100 ◦ C. However, Maia et al.18 reported
higher lectin activity levels than in the present study
in six cultivars of Brazilian cowpeas using trypsinated
blood cell treatment. These results are only generally
comparable with the present ones, because the authors
used rabbit blood cells (trypsinated rat, bovine, human
and hamster blood was used in the present study) and
no heat treatment was applied, which may affect lectin
affinity for carbohydrates in the blood cell membrane.
As mentioned above, no appreciable lectin activity
was observed in the PC, making it significantly
(P < 0.05) lower than in the WM. This is most likely
a result of the soaking process. Fern´andez-Quintela
et al.6 reported similar results in that they observed
no detectable lectin activity in protein concentrates
Table 4. Antinutritional factorsa (except trypsin inhibitors) in cowpea (Vigna unguiculata L. Walp var. IT86D-719) whole meal (WM), processed meal
(PM) and protein concentrate (PC)
Antinutritional factor
α-Amylase inhibitor
g equivalent kg−1 DM
g equivalent kg−1 protein
Lectin activity
HU kg−1 DM
g lectin equivalent kg−1 DM
Tannins
g tannic acid kg−1 DM
g tannic acid kg−1 protein
Phytates
g phytic acid kg−1 DM
g phytic acid kg−1 protein
Cyanogenic glucosides
g HCN kg−1 DM
WM
PM
PC
(8.4 ± 0.4) × 10−3 b
(3.4 ± 0.84) × 10−2 b
(1 ± 0.08) × 10−2 a
(4.8 ± 0.84) × 10−2 a
(6 ± 0.2) × 10−3 c
(8.1 ± 0.7) × 10−3 c
0.24 ± 0.04a
2.5 ± 0.41a
(1 ± 0.08) × 10−5 b
(6 ± 0.30) × 10−3 b
ND
ND
1.94 ± 0.04a
7.93 ± 0.16a
1.75 ± 0.02b
6.82 ± 0.06b
1.12 ± 0.02c
1.42 ± 0.02c
6.86 ± 0.01a
28.1 ± 0.09a
5.11 ± 0.02c
19.8 ± 0.53b
6.24 ± 0.02b
7.93 ± 0.020c
ND
ND
ND
a
Values are mean ± standard deviation of three determinations. Different letters in the same row indicate significantly different (P < 0.05) means.
DM, dry matter; HU, haemagglutinating activity unit; ND, not detected.
116
J Sci Food Agric 87:112–119 (2007)
DOI: 10.1002/jsfa
Bioactive factor content of cowpea raw meal and protein concentrate
from peas (Pisum sativum), faba bean (Vicia faba) and
soya bean, which they explained as being caused by
concentrate preparation; they used the same isoelectric
point method as in the present study.
Tannin was present in the WM, PM and PC fractions (Table 4). Tannin levels dropped progressively
as the meal was processed, producing significant differences among fractions whether expressed as g tannic
acid kg−1 DM or kg−1 protein. Ogun et al.4 reported
similar tannin content results for one raw cultivar
(Kano 1696) of the four cultivars they studied. After
processing (dehulling, cold soaking, hot soaking), no
tannins were detected in any of the four cultivars. Cold
soaking caused a non-significant decrease in tannins
in comparison with the raw grains, and hot soaking
led to a greater but still non-significant drop in tannins
in comparison with the raw grains. The tannin levels
in the present PM may have originated in tannins
from other parts of the grain besides the testa or from
residual hulls. If the latter is true, dehulling efficiency
was notably lower in the present study than in that of
Ogun et al.4
Despite slight variations in different studies,
dehulling and the protein concentration process clearly
affect tannin content in the final product. For example,
Fern´andez-Quintela et al.6 observed that antinutritional factor content, including tannins, drops after
protein concentrate preparation. Because the highest tannin concentrations in V. unguiculata and other
seeds are generally found in the testa,4,33 the lack of
detectable tannins in the PC probably resulted from
removal of the testa prior to protein extraction, as well
as retention of low-molecular-weight polyphenols34 in
the water-soluble (pH 4.3) phase.
There were significant differences (P < 0.05) in
phytate content among the WM, PM and PC
fractions on both a DM and protein basis (Table 4),
though each basis exhibited a different pattern.
The phytate concentration on a DM basis in the
PM was significantly lower (P < 0.05) than in the
WM and PC. When compared in terms of protein,
however, the concentration decreased progressively,
with significantly lower (P < 0.05) values between the
WM and PM and between the PM and PC. The
phytate concentration value (DM and protein basis)
in the WM was comparable to those found in many
other legumes.33,35,36 Hidvegi and Lasztity36 reported
that phytic acid content in cowpea meal can vary from
2.9 to 8.6 g kg−1 , while in soya protein concentrate
they reported a phytic acid content of 8.2 g kg−1 .
Oluwatosin29 found higher dry matter basis phytic
acid values in cowpeas than in the present study,
with values ranging from 25.07 to 16.05 g kg−1 . This
discrepancy may be explained by the fact that some
of their varieties, including var. IT86D-534 from the
same source as var. IT86D-719, resulted from recent
breeding programmes aimed at improving resistance
to pests and diseases.
Processing (i.e. dehulling and soaking) notably
affected phytic acid concentration in the present
J Sci Food Agric 87:112–119 (2007)
DOI: 10.1002/jsfa
study. Ogun et al.4 , however, reported that it had no
significant effect on phytic acid, with average values of
1.2 ± 0.02 g kg−1 with dehulling and 1.1 ± 0.02 g kg−1
with cold soaking for four cultivars; these are quite
different from the present results for the PM. This
contrast may be due to the extraction method or simply
to phytate solubility in var. IT86D-719. Comparison
of phytate concentration variation in terms of protein
content is not possible, since Ogun et al.4 did not
express their data on that basis.
On a protein basis the present PC had 3.5 times
less phytic acid than the WM and 2.5 times less
than the PM, with its value of 7.93 g kg−1 being
relatively close to the values of 9.03 g kg−1 for soya
bean isolate and 10.1 g kg−1 for protein concentrate
reported by Hidvegi and Lasztity.36 The difference
between the meals and the PC is probably due
to most of the phytates being in the cotyledon,
meaning that they would have been extracted at pH
9. However, the remaining phytates may be highly
water-soluble and therefore did not precipitate during
protein concentrate preparation. Phytic acid was not
completely eliminated in the PC, meaning that the
processing method did not remove it all and/or the raw
material had high concentrations. This results from the
phytic acid concentration in protein concentrates or
isolates being dependent on the raw materials from
which they are prepared as well as the processing
method. The final concentration may also be affected
by the protein–phytic acid interaction, which is highly
pH-dependent. A pH above a protein’s isoelectric
point can diminish the protein–phytic acid interaction,
while a pH below this point can raise the interaction,
though binding begins to decrease below pH 2.5.
No cyanogenic glucosides were detected in the
WM and thus they were absent in the PM and PC
fractions. This contrasts with the result of Colom´e
et al.,19 who found 2.9 mg HCN kg−1 in whole V.
unguiculata meal. The difference between the present
data and those of Colom´e et al.19 is probably due
to the fact that cyanogenic glucoside levels can vary
considerably depending on a crop’s location and
environmental conditions, as is the case with many
other antinutritional factor contents.
CONCLUSIONS
Overall, V. unguiculata L. Walp (IT86D-719) meal
appeared to contain only moderate or low levels
of potentially bioactive or antinutritional factors,
indicating that meals (WM and PM) from such
cowpeas grown in Yucatan, Mexico, or a protein
concentrate (PC) derived from them, are potentially
good foodstuffs. Given that neither the meals nor the
PC have high levels of adverse bioactive/antinutritional
factors, they can be effectively used as sources of
dietary protein, energy or essential amino acids with
only minimal processing. However, they cannot be
consumed as a sole protein source because they are
limited in sulphur amino acids and therefore must be
117
L Olivera-Castillo et al.
combined with cereals to balance their amino acid
profile.
Trypsin inhibitors, α-amylase inhibitor and lectins
in many legume seeds are albumin-like proteins
and thus soluble in aqueous solution. The low
concentrations of some antinutrients and the absence
of lectin observed in the cowpea PC (pH 4.3 insoluble)
would be consistent with the constituent bioactive
factors being albumin-like proteins. The persistence of
some trypsin inhibitor in the PC may be a carry-over
from its high concentration in the WM. Repeating
the solubilisation/precipitation procedures used here
with the PC may further reduce trypsin inhibitor
levels. Completely eliminating all antinutrients in
the PC, however, may not necessarily be the most
advantageous overall approach, since at certain levels
they can aid in disease prevention. In vivo nutritional
studies will be required to firmly establish these
properties and better understand this legume’s high
potential.
ACKNOWLEDGEMENTS
The authors thank C´esar Puerto-Castillo and Wilberth
´ for their laboratory assistance, Laura
Ch´e-Leon
Escobar-Brillones for help with the statistical analysis,
and Dr Luis Chel-Guerrero for facilitating the
tryptophan analysis. This research was done as
part of a British Council/Mexico HEL Program
(MXC/991/83). Support was also provided by the
Scottish Executive Environment and Rural Affairs
Department and the International Foundation for
Science (grant E/3171-1).
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