International Research Journal of Biotechnology (ISSN: 2141-5153) Vol. 2(8) pp. 192-197, November, 2011
Available online http://www.interesjournals.org/IRJOB
Copyright © 2011 International Research Journals
Full Length Research Paper
Effects of dehusking on the composition of
phytochemicals nutrients, antinutrients, minerals and
in-vitro multi enzyme digestibility of the seed of
Brazilian Jack beans (Canavalia braziliensis)
Oseni O. A1*. Ibeto A.U2 and Aruna M.O.3
1
Department of Medical Biochemistry, College of Medicine, Ekiti State University, Ado-Ekiti, Nigeria
2
Department of Chemistry, Federal University of Technology, Akure, Ondo State, Nigeria
3
Department of Biochemistry, Federal University of Technology, Akure, Ondo State, Nigeria.
Accepted 03 November, 2011
This study was aimed at assessing the phytochemicals, nutritional, anti-nutritional, minerals
composition and in-vitro multi enzyme digestibility of the whole and dehusked seed of Brazillian jack
beans Canavalia braziliensis popularly called kokondo in South Western Nigeria to ascertain its
industrial and nutritional potentials. There was no observable difference in the phytochemical
constituents as a result of dehusking; the percentage proximate composition of the whole and
dehusked seed were found to be total ash (7.5: 7.5) %; crude fiber (5.0: 2.3) %; crude protein (35.4: 31.5)
%; crude lipid (11.6: 11.0) % and carbohydrates (32.5: 41.2) % respectively. While the levels of the
antinutrient factors are tannins (0.41:0.49) mg/100TA; oxalate (6.48:4.6) mg/g; phytate (53.54: 35.46)
mg/g and cyanogenic glycosides (2.03:2.36) mg/kg. There was no significant deference in the macro and
micro mineral compositions as well as the in-vitro multi enzyme digestibility of the whole and dehusked
samples. This study revealed the nutritional profile of the seed as a good source of plant protein,
carbohydrates, basic phytochemicals, macro and micro minerals which are potentials that could be
exploited by food and pharmaceutical industries while dehusking caused a reduction in the levels of
some antinutrients.
Keyword: Canavalia braziliensis; Dehusking; Nutrient; Antinutrient; phytochemicals
INTRODUCTION
The Canavalia brasiliensis (Brazilian jack beans) is a new
world species with very wide distribution centres that
extends from Central America, Paraguay and Northeast
Brazil. It belongs to the family of leguminosae which is
probably the second most importantly source of food and
fodder next only to the graminesae-cereal grain (Alen and
Alen,1981).the legumes are good source of dietary
protein they are rich in lysine and tryptophan but low in
sulphur-containing amino acids like methionin and cystein
(Elegbede,1998). Legumes are cheaper than animal
products (meat, fish and egg) therefore; they are
consumed worldwide as major source of protein and
especially in the developing or poor countries where
*Corresponding author E-mail: ooseni2003@yahoo.com
consumption of animal proteins may be limited as a result
of economic, socio-cultural or religious factors (Onuorah
et al., 1989; Elegbede, 1998). Wild legumes are
important for food security, nutrition, agricultural
development and rotation of crops to improve the nation’s
economy. Many wild legumes are known for inexpensive
proteins, calorific value, essential amino acids, essential
fatty acids, fiber and vitamins. Antinutrients or
antinutritional factors may be defined as those
substances generated in natural feedstuffs by the normal
metabolism of species and by different mechanisms (for
example inactivation of some nutrients, diminution of the
digestive process or metabolic utilization of feed) which
exerts effect contrary to optimum nutrition. Being an
antinutritional factor is not an intrinsic characteristic of a
compound but depends upon the digestive process of the
ingesting animal (Akande et al., 2010). However, some
Oseni et al. 193
antinutrients may exert beneficial health effects at low
concentrations. The mechanisms by which adverse and
beneficial effects of food antinutrients operate are the
same. Canavalia brasiliensis has similarly been reported
to be underutilized worldwide; as a result, investigations
for functional food ingredients and nutraceutical products
are important in health promotion and disease risk
reduction. Though such foods which are expected to
render physiological benefits beyond their traditional
nutritional value also contain some phytochemicals,
hence the aim of this study to investigate the
phytochemials, nutritional, antinutritional potentials,
mineral and in-vitro multi enzyme digestibility of the seed
for animal and human benefits.
MATERIALS AND METHODS
Materials Collection
emulsion.
Test for alkaloids
5.0 mL of 1% aqueous hydrochloric acid was added
to2gm of each sample in a test tube, heated in a steam
bath and filtered; 1mL of the filtrate was treated with 6-10
drops of Dragendoff’s reagent. The presence of creamish
precipitate or turbidity after addition was taken for the
presence of alkaloid.
Anthraquinone Determination
5g of each of the sample was shaken with 10ml benzene,
filtered and 5ml of 10 percent ammonia solution added to
the filterate. The mixture was shaken and the presence of
a pink, red or violet colour in the ammoniacal (lower)
phase indicates the presence of free anthraquinones.
Methods
The dried pod of Canavalia brasiliensis was harvested
from a local farm at Federal University of Technology
Akure (FUTA) South gate in Akure Ondo State, Nigeria.
The pods were split and the seeds screened for defective
ones and then transported to the laboratory in a
polyethylene containers. The dried pods were divided into
two groups (whole seeds and dehusked seeds). All the
reagents and chemicals used in the study were of
analytical grade.
Test for phlobatannins
Deposition of a red precipitate when an aqueous extract
of each plant sample was boiled with 1% aqueous
hydrochloric acid was taken as evidence for the presence
of phlobatannins.
Teat for flavonoids
Phytochemical screening
Chemical tests were carried out on each extract to screen
for phytochemical constituents as described by Sofowora
(2006), Trease and Evans (1989) and Harborne (1984).
Test for tannins
0.5 g of the dried powdered samples was boiled in 20 ml
of water in a test tube and then filtered. A few drops of
0.1% ferric chloride was added and observed for
brownish green or a blue-black colouration.
Test for saponin
2.0 g of the powdered sample was boiled in 20 ml of
distilled water in a water bath and filtered. 10ml of the
filtrate was mixed with 5 ml of distilled water and shaken
vigorously for a stable persistent froth. The frothing was
mixed with 3 drops of olive oil and shaken
vigorously, then observed for the formation of
Three methods were used to determine the presence of
flavonoids in the plant sample.
5 mL of dilute ammonia solution was added to a
portion of the aqueous filtrate of each plant extract
followed by addition of concentrated H2SO4. A yellow
colouration observed in each extract indicated the
presence of flavonoids. The yellow colouration
disappeared on standing.
Few drops of 1% aluminium solution were added to a
portion of each filtrate. A yellow colouration was observed
indicating the presence of flavonoids.
A portion of the powdered plant sample was in each
case heated with 10 mL of ethyl acetate over a steam
bath for 3 min. The mixture was filtered and 4 mL of the
filtrate was shaken with 1 mL of dilute ammonia solution.
A yellow colouration was observed indicating a positive
test for flavonoids.
Salkowski test for cardiac glycosides (steroidal ring
or terpenoids)
5.0mL of each extract was mixed in 2mL of chloroform,
194 Int. Res. J. Microbiol.
and concentrated H2S04 (3mL) was carefully added to
form a lower layer. A reddish brown colouration of the
inter face was formed to show positive results for the
presence of terpenoids.
1.0ml of 5% potassium iodide solution added to the
solution which was titrated against 0.01M silver nitrate
solution, the blank was also titrated as well until the end
point was indicated by a faint but permanent turbidity.
Keller-Killani
(deoxysugar)
Phytate Content
test
for
cardiac
glycosides
50mL of each extracts was treated with 2mL of glacial
acetic acid containing one drop of ferric chloride solution.
This was under layered with 1.0mL of concentrated
sulphuric acid. A brown ring at the interface indicates a
deoxysugar characteristic of cardenolides. A violet ring
may appear below the brown ring, while in the acetic acid
layer, a greenish ring may form just gradually throughout
thin layer.
Phytate content was determined according to the
Wheeler and Ferrel (1971). 4g of sample was soaked in
100mL of 2% HCl solution for three hours and filtered
through Whatman number 2 filter paper. 25mL of the
filtrates was placed in a conical flask and 5ml of 0.3%
ammonium thiocyanate solution was added, after which
53.5ml of distilled water was added. The solution was
titrated against a standard iron III Chloride solution
containing 0.00195g of iron per mililiter until a brownish
yellow colour persists for five minutes. The Phytate
content was expressed as mg/g phytate in the sample.
Lieberman’s Test for steroidal nucleus
2.0mL of acetic anhydride was added to 0.5 g of each
sample with 2.0mL H2SO4. The colour changed from
violet to blue or green in some samples indicating the
presence of steroids.
Determination of proximate composition
Crude protein (N x 6.25), crude fat, crude fiber, ash and
moisture contents of both whole and dehusked seeds
were determined according to the standard methods of
Association of Official Analytical Chemists (AOAC 1990).
Total carbohydrate was calculated based on Müller and
Tobin (1980): Total carbohydrates (%) = 100 – (crude
protein + crude lipid + crude fiber + ash) (%).
Oxalate content
The oxalate content of the samples was estimated
according to the procedure of Day and Underwood
(1986). 1g of each of the sample was weighed and 75ml
of 1.5N H2SO4 solution was added, the mixture was
carefully stirred intermittently with a magnetic stirrer for
one hour and then filtered using whatman No 1 filter
paper. 25ml of the filtrate was collected and titrated hot
(80 – 90oC) against 0.1N KMnO4 solution till the end point
of a faint pink colour appears that persist for at lest 30
minutes. Then the amount of oxalate in each sample was
estimated and expressed in mg/g oxalate.
Tannin
Quantitative determination of antinutrients
Cyanide Content
The method used for the determination of the cyanide
was the one from AOAC (1990). 4g of each sample was
soaked in a mixture containing 40ml of distilled water and
2ml of orthophosphoric acid. It was then mixed,
stoppered and left overnight at room temperature to set
free all the bounded hydrocyanic acid. The resulting
sample was transferred into distillation flasks and a drop
of paraffin was added as antifoaming agent together with
broken chips as antibumps. The flask was filled to other
distillation apparatus and distilled about 45ml of the
distillate into a receiving flask that contained 40ml of
distilled water containing 0.1g of NaOH pellets. The
distillate was then transferred into 50ml volumetric flask
and made up to mark with distilled water. 20ml of the
distillate was collected and place in the conical flask with
The spectrophotometric determination of tannin was done
according to the method of Makkar et al., 1993) by
extracting 0.2g of the powered seeds in 70% acetone.
0.5ml of the sample extract aliquots was mixed with equal
volume of water and 2.5ml folin-cioalteau reagent was
added as 2.0ml of 10% sodium carbonate was
subsequently added to the reaction mixture which was
incubated at room temperature for 40 minutes and the
absorbance was measured at 700nm using a
spectrophotometer. The total tannin was express as
mgTA equivalent/100g of the sample.
Determination of mineral composition
The mineral elements were determined after wet
digestion of samples with concentrated H2SO4, using
Atomic Absorption Spectrophotometer (Buck Scientific,
East Norwalk, CT, USA). All determinations were carried
Oseni et al. 195
Table 1. Phytochemical constituents of Canavalia brasiliensis seed
Alkaloids
Cyanogenic glycoside
Flavonoids
Oxalate
Phlobatannins
Phytate
Saponins
Steroids
Tannins
Terpenoids
Whole seed
+
+
+
+
+
-
Dehusked seed
+
+
+
+
+
-
(+) Sign indicates the presence of the constituents
(-) Sign indicates the absence of the constituents
Table 2. Proximate Composition of C. brasiliansis
Proximate Parameter
Moisture content
Total ash
Crude fibre
Crude protein
Crude lipid
Crude carbohydrates (by difference)
% Composition in Samples
Whole Seeds
Dehusked Seeds
8.0
6.5
7.5
7.5
5.0
2.3
35.4
31.5
11.6
11.0
32.5
41.2
Table 3. Antinutrient content of Canavalia brasiliensis seed
Sample
Whole seed
Dehusked seed
Tannin mg/100g TA
0.41
0.49
Phytate mg/gPA
53.54
35.85
out in triplicates.
In-vitro multi enzyme protein digestibility
The method of Hsu et al., (1997) was employed where
three enzymes; trypsin (EC 3.4.2.145 PN;
TI 005) Chymotrypsin (EC 3.4.21.1; PN, C4129) and
peptidase (EC 3.4.23; 1; PN P6887) were used. A 50mL
of each of the sample suspension containing 6.25mg/mL
of distilled water was prepared. These were adjusted to
pH 8 using 0.1M HCl or 0.1M NaOH and kept in a water
0
bath and maintained at 37 C. The enzyme solution
containing 1.6mg trypsin, 3.1mg of chymotrypsin and
1.3mg of peptidase per mL of distilled water was
prepared and maintained at ice bath after which its pH
has been adjusted to 8.0. 5.0mL of the enzyme solution
was added to each of the sample suspension maintained
at 370C in the water, mixed and allowed to stand with
constant stirring. Their pH was recorded after 10 minutes
respectively.
The percentage digestibility of each
Oxalate mg/g
6.48
4.86
Cyanide mg/kg
2.03
2.36
sample was calculated using the regression equation
method of Hsu et. al., (1997).
Y = 210.464 – 18.103X. Where Y = % in vitro
digestibility, X = pH of sample after 10 minutes.
RESULTS AND DISCUSSIONS
Discussion
The phytchemical screening (Table 1) and quantitative
estimation of the antinutrient components (Table 3) of the
Canavalia brasiliensis showed the presence of saponins,
phytate, oxalate, tannin and alkaloids. The seeds of
Canavalia brasiliensis were very high in phytate and has
lower amount of oxalate in both samples (whole seed and
dehusked seed). The percentage of phytate in whole
seed was observed very high due to the presence of the
epicarp in samples as shown in Table 3. The result is in
line with work of other researchers. The presence of high
amount of phytate has been reported by Isidro et al.,
196 Int. Res. J. Microbiol.
Table 4. Mineral Composition of C. brasiliensis
Minerals
Macro minerals
1
Ca
2
Na
3
K
4
5
6
7
8
Mg
Mn
Zn
Fe
Cu
Concentration in Samples (mg/100g)
Whole seed Dehusked seed
16.10
23.40
49.48
42.76
26.60
24.26
Micro minerals
20.92
25.11
0.28
0.46
0.14
0.10
0.2
0.3
0.04
0.04
Table 5. In-vitro Multi-Enzyme Digestibility of C. brasiliensis
Sample
Whole seeds
Dehusked seeds
% digestibility (10 minutes)
71.81
72.18
(2001). This is a major factor that implicated the
Canavalia brasiliensis seeds as containing strong
antinutrients. The factor is capable of binding and
preventing the absorption of protein and can chelate
3+
2+
2+
divalent and trivalent cations such as Fe Ca , and Mg
(Barcellos et al 1993). The amount of phytate was found
in large quantity in the whole seed with the value of
53.5mg/g, this was reduced to almost 36mg/g by
dehusking of the seed.
The proximate analysis of Canavalia brasiliensis seeds
revealed that the seed contains 35.4% crude protein in
whole seed and 31.5% in dehusked seed. this was in line
with the report of Gomes et al (1998), who reported
31.9% to 41.6% (Mayworm et al., 1998) crude protein,
with some variance to the report of Mayworm et al.
(1988) and Gomes et al., (1988) who reported a
percentage crude protein of 41.6%, 52.3% carbohydrate,
12.3% crude fibre, 2.8% ash and 1.2% oil. There are
some major differences in the percentage composition of
the proximate components. The whole seed has higher
amount of moisture, crude fibre, crude protein, and lipid.
The two samples have the same amount of ash content.
The amount of digestible carbohydrate in dehusked seed
is greater than that of whole sample; this was because
the epicarp of the whole seed has caused an increase in
crude fibre. Since the total carbohydrate is obtain by
subtraction of the sum of the Moisture, Ash, Protein,
Crude Fibre and Fat from the total weight of the sample
from 100 percent.
The results of mineral composition in Canavalia
brasiliensis seeds was similar to other tropical forage
legumes as reported by (Alverenga et al., 1995, Cobo et
al., 2002) with the exception of the high Ca content of
16.1mg/100g in whole seed and 23.4mg/100g in
dehusked seed (Table 4), this could be another possible
reason for the presence of some structural carbohydrate
such as lignin, cellulose, hemicelluloses. This may also
add to the increase in the ash and crude fibre content, as
it could also lead to decrease in the total amount of
carbohydrate in whole seeds.
In Table 5, the digestibility of raw whole seeds and
dehusked seeds after 10 minutes digestion were
compared. The digestibility of dehusked seeds was better
(72.81%) than that of the whole seeds. It shows that
dehusking the seeds improves the digestibility of the
seed. This is in support of the report by Osagie (1998)
that dehusking is a simple processing method for
reducing the contents of some compounds that are highly
associated with the seed coat in food and hence improve
protein digestibility. Similaly the low digestibility recorded
for the whole seed could be as a result of presence of
phytic acid as reported by Osagie (1998) that phytate
affects digestibility of proteins by chelating Ca or binding
with substrate or proteolytic enzymes. This result can be
confirmed by the low concentration of Ca in the whole
seed compared to that of the dehusked seed. Also, the
low digestibility of the whole seed which has higher fibre
content support the research by Blackburn and
Southgate (1981) that the high fibre diet result in a
statistically significant reduction in Nitrogen digestibility of
approximately 9% compared to low fibre diet.
CONCLUSION
We believed that the raw seeds of Canavalia brasiliensis
were either toxic or valueless to human, but the results of
Oseni et al. 197
this work have shown that Canavalia brasiliensis seeds
are important as nutritious diet. It could also be used to
formulate animal feeds. Though there is still need for
further research on its toxicity considering the high crude
protein content of the seeds which is a nutrition factor
indicative of its high potential as protein supplement in
human diets. However, this variety of legume is tedious
to process for food, hard to cook and contain high levels
of anti nutritional factors and unacceptable flavor which
must be removed without sacrificing taste or other
nutritional factors for the plant to be acceptable as food.
Therefore, dehusking and other processes could help to
reduce some of these antinutrients and improve the
nutritional status.
REFERENCES
Akande FB, Adejumo OA, Adamade CA, Bodunde J (2010). Processing
of locust bean fruits: Challenges and prospects. Afr. J.Agric. Res.
5(17):2268-2271
Allen ON, Allen CK (1981). The Leguminosae. A source book of
characteristics, uses and nodulation. Macmillan Publishers Ltd.,
London, U.K.
Alvarenga RC, Costa LM, da Moura Filho W, Regazzi AJ (1995).
Características de alguns adubos verdes de interesse para a
conservação e recuperação de solos. Pesquisa Agropecuária
Brasileira 30(2): 175-185.
AOAC (1990) Official methods of analysis. 15th edition Association of
Official Analytical Chemistry. Washington D.C.
Blackburn NA, Southgate DAT (1981). Protein Digestibility and
Absorption: Effects of Fibre, and the extent of individual variation.
ESN: FAO/WHO/UNUEPR/81/32
Barcellos GBS, Almeida LM, Moreira RA, Cavada BS, Oliveira JTA, de
Carlini CR (1993). Canatoxin-, concanavalin A- and canavalin-crossreactive materials during maturation of Canavalia brasiliensis (Mart.)
seeds. Planta 189: 397- 402.
Cobo JG, Barrios E, Kass DCL, Thomas R (2002). Decomposition and
nutrient release by green manures in a tropical hillside
agroecosystem. Plant and Soil 240: 331-342.
th
Day RA Jr,Underwood AL (1986). Quantitative Analysis, 5 Ed. Prentice
– Hall Publication, pp. 701
Elegbede JA (1998). Legumes In: Osagie AU and Eka OU (Eds),
“Nutritional Quality of Plant
Foods. Post Harvest Research Unit, Department of Biochemistry,
University of Benin, Benin City, Nigeria. Pp. 120-133.
Gomes JC, Epstein M, Maffia LM, Sant’ Anna R (1988). Composicao
quimica de sementes do feijao-bravo e de seu isolado proteico.
Arquivos de Biologiae Technologia 31: 443-459.
Mayworm MAS, Serra do Nascimento A, Salatino A (1998). Seeds of
species from the caatinga: proteins, oils and fatty acid contents.
Revista Brasileira de Botânica 21:299-303.
Makkar HPS, Blummel M, Borowy NK, Becker K (1993). Gravimetric
determination of tannins and their correlations with chemical and
protein precipitation methods. J. Sci. Food Agric., 61: 161-165.
Müller HG, Tobin G (1980). Nutrition and Food Processing. Croom
Helm Ltd., London.
nd
Harbone JB (1984 ). Phytochemical method 2
edition London.
Chapman and Hall, Ltd. ELBS pp. 12-56
Hsu WW, Vavak DL, Setterlee LD, Miller GA (1977). A multienzyme
technique for estimating protein digestibility. J. Food Sci. 42: 12691273.
Isidro R, Montenegro de Sales FJ, Cavada BS, Grangeiro TB, Moreira
RA (2001). Acão de lectina de sementes de Canavalia brasiliensis
Mart. sobre o comportamento da saúva do nordeste (Atta opaciceps
Borgmeier, 1939). Revista de la Facultad de Agronomía, Universidad
Central de Venezuela 27: 77-86.
Onuorah VI, Uwaegbute AC, Enwere NJ (1989). Cowpea utilization and
(Practical). In Food crops production, utilization and nutrition. Mbah,
M.B and D.O Nnayelugo (Eds). Dotam Publications Ltd, Ibadan. Pp.
121-126
Osagie AU (1998). Antinutritional factors. In: Osagie AU and Eka OU
(eds), Nutritional Quality of Plant Foods. Nigeria: Post Harvest
Research Unit, Department of Biochemistry,University of Benin,
Benin City, pp: 120-133. ISBN: 978-2120-02-2.
Sofowora A (2006). Medicinal plant and traditional medicine in Africa
Spectrum
Book Limited Ibadan Nigeria.
Trease GE, Evans WC (1989). Pharmacognosy 12th Ed. Bailliere
Tiindal pp 194-195.
Wheeler EL, Ferrel RA (1971). A method for phytic acid determination in
wheat and wheat flour. Cereal Chem., 48: 313-314.