Journal of Food Composition and Analysis 1 (2014) 1−10
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fliF
OOD
ANALYSI
S
Physico-chemical properties and urease activity of red kidney bean
(Phaseolus vulgaris L.) flour
Cyril John A. Domingo
Masters of Science in Food Science, Graduate School, University of the Philippines Los Baños, 4031, Philippines.
Accepted 17 February 2014
_____________________________________________________________________________
Abstract
Physico-chemical properties and urease activity of red kidney bean flour were investigated. Moisture
content, ash and TSS were 12.31 % ± 0.12, 3.84 % ± 0.28 and 2.73 °Brix ± 0.12, respectively. The pH of
the sample is 6.7, this value is near neutrality. The value for parameters L*, a* and b* were 87.86, 0.17
and 12.82, respectively. This can be associated to the light color of the flour with green and red hue. Low
urease activity of 0.2 was also obtained. The phospholipids were analyzed by thin-layer chromatography
and found that red kidney bean is comprised of phosphatidyl serine, lysophosphatidyl ethanol amine and
two unknown phospholipids which were not identified due to the limited reference Rf values. Gas
chromatography was employed on the determination of fatty acid content of the food sample. Results
revealed that red kidney bean oil contained moderate to high level of saturated fatty acid in the form of
stearic acid (16.50%) and unsaturated fatty acids probably in the form of oleic or linoleic (83.54%). It is
very important to know these physico-chemical properties for these dictates a lot in terms of the
processing of the sample commodity.
Keywords: red kidney bean, compositional analysis, phospholipid, fatty acid, chromatography
______________________________________________________________________________
I. Introduction
Common dry bean (Phaseolus vulgaris L.)
is one of the most important crops of the
world with different physical, biochemical
and sensory properties. The global
production of the dry beans in 2010 was
22.9 million metric tonnes and five leading
producers were India, Brazil, Myanmar,
United States and Mexico (FAO, 2012).
The different classes of common dry bean
include black bean, cranberry bean, great
northern bean, kidney bean, navy bean,
pinto bean and small red bean. Kidney beans
are good source of protein, starch and
dietary fibres (Osorio-Diaz et al., 2003).
Physico-chemical properties of kidney beans
change with postharvest handling and
storage conditions resulting in a reduction in
their cooking, eating and nutritional quality
as well as consumer acceptance (Njintang et
al., 2001).
Physico-chemical properties of food
products are analyzed for various reasons
such as compliance with legal labeling
requirements, assessment of product quality,
C.J.Domingo / Journal of Food Composition and Analysis 1 (2014) 1−10
and determination of nutritive value,
detection of adulteration, research and
development. Knowledge of the moisture
content is often necessary to predict the
behavior of foods during processing while
ash content provides a measure of the total
amount of minerals within a food. Color and
pH serves as determinants to food product
quality (Nielsen, 2010).
Legumes are known to contain urease - an
enzyme which acts on non-peptide C-N
bones in linear amides and having been
known to convert urea to ammonia and CO2
(Teng et. al, 1988). Its inactivation is
important in certain feedstuffs fortified with
urea for ruminants. Also, the loss of urease
activity indicates an inactivation of other
enzymes. Most importantly, it is used as one
of the quality control parameter in some
food products.
Oil of legume seeds are mainly constituted
by fatty alcohols, wax esters, hydrocarbons,
tocopherols, phenolic compounds, volatiles,
pigments, phospholipids and triterpenic
acids. The ability of an analytical method to
characterize a vegetable oil is based on the
identification and quantification of those
compounds that are expected to be in
connection with their origin and quality
attributes (Cert et al., 2000).
Phospholipids
(PLs)
are
important
constituents of oilseeds. The measurement
of PLs is important in determining the
stability and quality of vegetable oils.
Phospholipids are undesirable in oil since
they are responsible for oil discoloration
during deodorization and steam distillation
and losses of neutral lipids during
neutralization. They affect the stability of
the oil by chelating metals and increasing
the amount of metal ions. The removal of
PLs results in elimination of iron and
copper, which increases the oxidative
2
stability and facilitates the refining process
(Cert et al., 2000).
The separation and identification of PLs can
be made by thin-layer chromatography
(TLC) (IUPAC, 1987).
The analysis of fatty acid methyl esters
(FAMEs) is used for the characterization of
the lipid fraction in foods and is one of the
most important applications in food analysis.
The
qualitative
and
quantitative
determination of fatty acid constituents is
often done by gas chromatography (GC).
GC in general assumes that the compounds
injected are volatile at the temperature of
analysis and that they do not decompose at
either the temperature of injection or
analysis. In standardized analytical methods,
flame ionization detection (FID) is the most
widely used. Mass spectrometry (MS)
allows obtaining molecular mass data,
structural information and identification of
compounds (Cert et al., 2000).
The major aim of this study is to evaluate
and characterize the biochemical and
physical properties of red kidney bean flour.
More specifically, to evaluate the moisture
content, ash, pH, color, urease activity,
phospholipids and fatty acids composition of
red kidney bean flour.
II. Materials and Methods
2.1 Chemicals and Reagents
All solvents and chemicals used were
analytical-reagent grade. The red kidney
bean seeds were obtained from the local
market of Los Baños, Laguna.
C.J.Domingo / Journal of Food Composition and Analysis 1 (2014) 1−10
2.2 Preparation of Sample
The red kidney bean seeds were manually
dehulled and were ground using osterizer.
One hundred grams of the ground bean was
sieved through 20-mesh screen and stored in
airtight container at refrigerated temperature
until used. Ten grams of 20 mesh powder
went through a four times extraction
processes by 40ml petroleum ether. The
samples after extraction were concentrated
using the rotary evaporator (Yamato rotary
evaporator model RE-46) and placed in a
vial at freezing temperature until used.
2.3 Physico-chemical Properties of Red
Bean Flour
3
crucibles was less than 0.001 g. The lost
weight is calculated as the moisture of the
sample.
% MC =
Minitial – Mfinal x 100
Minitial
The sample from moisture content
determination was heated in the furnace for
five hours at 550°C. Ash content was
derived by dividing the weight of the
remaining residue over the original weight
of sample.
% Ash =
Weight of residue x 100
wt. of sample
2.4. Urease Activity
2.3.1 Color, total soluble solids and pH
value measurement
The surface color of flour was measured
using chromameter (CAPSURE). The
parameters recorded were *L, *a and *b
coordinates. The bean flour was diluted with
distilled water at a ratio of 1:10
(sample:water) and then, pH was measured
using calibrated pH pen (Milwaukee pH
600) and total soluble solids reading was
done using a refractomer.
2.3.2 Compositional assessment
The compositional analysis of the bean flour
included the determination of moisture
content using oven drying method
(Memmert Convection Incubator). Dried
samples (0.2-0.3 g) were placed in a tared
crucible and oven-dried at 105°C overnight.
The sample was then allowed to cool at
room temperature for 30 minutes in a
desiccator. Weights were afterwards
recorded and the process of heating and
weighing was redone for another 30
minutes. The process was repeated until the
difference in the weight of the cooled
Approximately 0.20g of flour sample was
weighed in into a test tube and was added
with 10mL of buffered urea solutions. The
samples were mixed and incubated at 30°C
for 30 minutes. The blank was prepared by
weighing the same amount of sample into a
test tube and was only added with 10ml
phosphate buffer solution. It was mixed and
incubated along with the samples but with a
5 minute interval. The pH of the supernatant
liquid was determined at exactly 5 minutes
after removal from the bath. The difference
between the pH of the test and pH of the
blank was used as the index of urease
activity.
2.5 Phospholipid Profile
2.5.1 Extraction of phospholipid
Phospholipid extraction was carried out
using 5g of deffated sample with 100 ml
choloform: methanol (2:1) in warring
blender as solvent. After filtering, the filtrate
was concentrated using a rotary evaporator.
One gram of oil sample was dissolved in 4
ml chloroform.
C.J.Domingo / Journal of Food Composition and Analysis 1 (2014) 1−10
2.5.2 Thin-layer chromatography
Commercial TLC glass sheets coated with
silica gel were used in this exercise.
4
increases in prolonged standing of the
plates. If no color development was
observed, pre-heat the plates at 100oC for 5
minutes before spraying the solution.
2.6 Fatty Acid Profile
The developing solvent was composed of
170:25:25:4 (v/v) of chloroform: methanol:
acetic acid: water. One ul of extract was
spotted on the plate. Then, the spotted plate
was placed in the developing chamber, and
then dried.
2.5.3 Detection of phospholipids
Spots were first made visible using iodine
vapor. Dried TLC plates were placed inside
the reaction chamber (containing iodine
vapor) and spots development were
observed in about a few minutes. The brown
spots seen were temporary and were
encircled softly using a pencil as marker.
The markers were then used as a guide to
facilitate spraying of other reagents that
would make the spots completely visible.
For the spot development of amino
phospholipids, the plates were heated at 100
°C prior to spraying with the ninhydrin
solution. If no perceived spot development
was observed on probable phosphatidyl
ethanolamine and phosphatidyl serine, then
the plates were reheated in oven at 100°C
for 15-20 minutes. Development of pink
spot was monitored.
TLC plates were sprayed with 20%
perchloric acid and then heated to 100°C for
5 minutes. If no spot development was
observed the plates were reheated at 100°C
for 15- 20 minutes. Spot development is
gray or black.
For
phosphorous
containing
lipid
compounds (containing phosphate group),
blue spots on white or blue-gray background
are indicative results. Color intensity
2.6.1 Esterification
Approximately 0.02 grams of oil sample
was weighed in a screw capped tube, then
added with 4 ml methanolic HCl and then
mixed using a vortex mixer. The mixture
was incubated in an oven at 50°C for 10
hours, allowed to cool at room temperature
and was added with 5 ml hexane layer to
extract the ether vigorously. The two layers
were then allowed to separate. The hexane
layer contains fatty acids methyl ester and
was used in the chromatography analysis.
2.6.2 Gas chromatography
Roughly one μl of fatty acid methyl ester
sample was injected into the HewlettPackard Model 5890A Gas Chromatogram
equipped with a hydrogen flame ionization
detector under the following conditions: (1.)
column, 6ft x 1/8 in. stainless steel tube
column packed with 15% diethylene
glycosuccinate (DEGS) in Chromosorb W
(80/100mesh), (2.) carrier gas, nitrogen (20
ml/minute), (3.) injection temperature at
200°C, (4.) column temperature at 180°C,
(5.) hydrogen and air at 10 and 50 psi
respectively and (6.) the recorder, HewlettPackard Model 3396 Integrator. Each
chromatogram was allowed to run for 30
minutes.
The integrator calculates the amount of fatty
acids in the sample and expressed as
percentage. A standard mixture of 1 µl was
injected in the gas chromatography. Equal
amounts of methyl esters were present in the
standard (i.e. linoleic, linolenic, palmitic
stearic and etc.).
C.J.Domingo / Journal of Food Composition and Analysis 1 (2014) 1−10
III. Results and Discussion
Proximate analysis is an important criterion
to assess the overall composition and
nutritional status of any ingredient intended
for food use (Qayyum et al., 2012). The
results of the pysico-chemical analysis of the
red kidney bean are presented in Table 1.
The moisture content and ash were 12.31 %
± 0.12 and 3.84 % ± 0.28. Present findings
are in conformity with values described in
previous
literature;
however,
slight
variations may be due to varietal differences
and environmental conditions. Wani et al.
(2013) found that moisture content and ash
were 10.40 % ± 0.44 and 3.5 % ± 0.04,
correspondingly.
The data pertaining to present study are
closely related with the work of Sasanam et
al. (2011), they delineated 12.39 % ± 4.60
and 3.90 % ± 0.12 moisture and ash
contents, respectively.
The pH which is the negative logarithmic
function of the hydronium ions present in
the sample affects the flavor and microbial
spoilage of the food (Nielsen, 2010). Acidic
environments suggests less or inhibited
growth of microorganisms while pH close to
neutral makes the food product prone to
microbial contamination. The pH value of
the sample is recorder at 6.7. This means
that the food product requires processing
5
under pressure (higher than 100°C). The pH
obtained in the study is quite high compared
to the approximate pH reported by USFDA
(2007) which ranges from 5.40 to 6.0. The
total soluble solids (TSS) of the sample is
recorded at 2.73 °Brix ± 0.12 this result is in
agreement to the reported value of USDA
(2013) which is 2.10 °Brix.
Red kidney bean flour color was
characterized by the parameters L*, a* and
b* using a CAPSURE chromameter. L*
indicates brightness (0 = black → +100 =
white), a* indicates redness (-60 = green →
+60 = red), b* indicates yellowness (-60 =
blue → +60 = yellow) and all three axis
intersect at their mid points to form a sphere
from which any color can be plotted (Wood
et al., 2012).
Data collected for L*
parameter value is 87.86 which is close to
100 which corresponds to the flour’s beige
color. Low a* value (0.17) and high b*
value (12.82) were obtained indicating its
greener and redder hue, respectively. The
data is in agreement with the results of Wani
et al. (2013).
Urease and tripsin inhibitor are proteolyticinhibiting substances which pose a negative
impact growth of farm and industrially
grown animals since it counters the function
of trypsin (digestive enzyme) (Mcnaughton,
1981).
Table 1. Physico-chemical properties and urease activity of red kidney bean powder.
Sample:
Parameters
Red kidney
Moisture,
Ash, (%)
pH
TSS,
Color
Urease
bean
(%)
(°Brix)
activity (ΔpH)
L* 87.86
6.7
2.73 ± 0.12
a* 0.17
0.2
This study 12.31 ± 0.12 3.84 ± 0.28
b* 12.82
Sasanam et
12.39 ± 4.60 3.90 ± 0.12
al. (2011)
L* 81.6
Wani et al.
10.40 ± 0.44 3.5 ± 0.04
a* 1.3
(2009)
b* 7.9
C.J.Domingo / Journal of Food Composition and Analysis 1 (2014) 1−10
Urease exhibits the same behavior to trypsin
inhibitor deactivation; thus, showing a great
potential as an index of the safety of a food
material (Mcnaughton, 1981).
When the substrate urea is present, urease
breaks it down into carbon dioxide and
ammonia. Since ammonia is a basic
compound, its presence raises the pH of the
solution. Red kidney bean carried a urease
activity of 0.2. The author had not found any
relevant information on the urease activity
of the said sample. Thus, concluding red
kidney bean has reasonably small or absent
urease activity.
In array of investigations, variations in
proximate composition of red kidney bean
flour had been observed owing to different
environments, genotype and analytical
methods (Qayyum et al., 2012).
Dry beans contain 1 to 3% lipid depending
upon the species, origin, location, climate,
season, other environmental conditions, and
type of soil on which they are grown. Lipids
in beans are made up of triglycerides,
accompanied by smaller proportions of free
fatty acids, sterols, and sterols esters,
phospholipids and glycolipids (Siddig and
Uebersax, 2012).
Various tests were conducted in order to
determine the phospholipids content of red
kidney bean oil. Different spray reagents
were used in which some were specific for a
6
particular functional group. Positive results
can indicate the presence of a certain
phospholipid. Also, the retention factor of
each spot that developed for each sample
was also determined. In comparing the
experimental Rf values with the standard, a
credible phospholipid identity for the sample
in parallel with the results of some other
tests can be establish.
B
A
C
U
U
U
U
U
U
PS
PS
U
U
LP
LP
PS
Origin
Figure 1. Thin-layer chromatography of
phospholipids from red kidney bean oil
viewed in various visualizing agents.
(A=Ninhydrin,
B=Perchloric
and
C=Molybdenum; PS=Phosphatidyl serine,
LP=Lysophosphatidyl and U=Unknown)
Table 2. The Rf values and the spray reaction of the different class of phospholipids present in
red kidney bean oil.
Visualizing agents
Identity of
Phospholipid
Possible
Iodine
Perchloric
Ninhydrin Molybdedum
No.
Phospholipids
1
0.04
0.06
0.04
Phosphatidyl serine
2
0.18
0.19
Lysophosphatidyl
ethanol amine
3
0.46
0.46
0.47
Unknown
4
0.70
0.76
Uknown
C.J.Domingo / Journal of Food Composition and Analysis 1 (2014) 1−10
Table 2 shows that phospholipid no. 1, 2 and
3 are positive to iodine test. Generally, all
phospholipids present in red kidney bean oil
should yield positive result in iodine.
According to (Nelson and Cox, 2004),
iodine reacts reversible with the double
bonds in fatty acids, such that lipids
containing unsaturated fatty acids develop a
yellow or brown color. Molybdenum spray
on the other hand is for the detection of
phosphate ester containing compounds. It is
therefore expected that no. 1, 2, 3 and 4
would be positive for molybdenum reaction
because of the presence of the phosphate
group in the hydrophilic head of
phospholipids. However, phospholipid no. 1
gave a negative result for this test. This may
due to the fact that though ammonium
molybdate spray has long been used for the
detection of phospholipids, its result is not
satisfactory when thin layer silica gel plates
are used and needs a longer time for
development of color (Goswami and Frey
1971). Phospholipid no. 1 got a positive
result for ninhydrin test, an indication of the
presence of free amino group. Based on the
Rf value obtained in Table 1, no. 1 is
positively identified as phosphatidyl serine
because it is close to the Rf value of 0.06.
Meanwhile, phospholipid no. 2 with an Rf
value of 0.18 and had a positive result on
iodine and molybdenum tests is believed to
be lysophosphatidyl ethanol amine. This
compound contains a free amino group
which yields a positive result (red-violet
7
shown in Fig. 1. However, the contrary was
obtained which may due to the low
concentration of the phospholipid present in
the sample to effect a positive reaction on
ninhydrin test. Lastly, phospholipid no. 3
and 4 were not identified because there were
no literature Rf equivalents for both
compounds. From the data presented it can
be deduced that both compounds consist of
free amino group because they yield a
positive result in ninhydrin test. Further tests
are therefore required to confirm the identity
of phospholipid no. 3 and 4.
The measurement of PLs is important in
determining the stability and quality of
vegetable
oils.
Phospholipids
are
undesirable in the oil since they are
responsible for oil discoloration during
deodorization and steam distillation. They
affect the stability of the oil by chelating
metal ions, therefore increasing oxidative
processes (Arruda and Dimick, 1991).
Lastly, the fatty acid profile of red kidney
bean oil is presented in Table 3. According
to the data of Siddig and Uebersax (2012),
the major fatty acids of red kidney beans are
linoleic and linolenic acids corresponding to
a concentration of about 26.9% and 50.6%,
respectively. This constitutes to about 85.8%
total unsaturated fatty acids present in red
kidney bean.
Table 3. Retention time and percent concentration of fatty acids in red kidney bean oil.
Standard
Red kidney bean oil
Identity (ME
Retention
Retention time,
% Conc. (Siddiq
% Conc.
form)
time, Rt (min)
Rt (min)
and Uebersax, 2012) (This study)
Palmitic, C16
18.04
13.4
Stearic, C18
21.44
21.93
0.74
16.50
Oleic, C18:1
24.61
22.73
8.3
83.54
Linoleic, C18:2
26.9
Linolenic, C18:3
27.56
50.6
Arachidic, C20
29.26
spots) when sprayed with ninhydrin as
C.J.Domingo / Journal of Food Composition and Analysis 1 (2014) 1−10
8
Other fatty acids components of red kidney
bean such as stearic and palmitic acid which
constitutes to about 8.3% total saturated
fatty acids.
Figure 2. Chromatogram of fatty acid methyl
ester (FAME) standards that were used. Peaks
from left to right: Hexane, Palmitic Acid ME
(C16), Stearic Acid ME (C18), Oleic Acid ME
(C18:1), Linolenic Acid ME (C18:3) and
Arachidic Acid ME (C20).
Comparison of the relative retention time of
an unknown component with that of a
reference compound analyzed under the
same conditions and related to the same
standard makes the identification of the
component
more
feasible.
The
chromatogram of red kidney bean sample
(Fig. 3) generated two peaks with retention
times of 21.93 (peak 1) and 22.73 (peak 2)
corresponding to concentrations of 16.50%
and 83.54%, respectively. Comparing with
the standards (Fig. 2), peak 1 was probably
stearic acid while peak 2 may be oleic or
linoleic acid. Retention times frequently
vary slightly from one analysis to another,
so in the direct comparison of the retention
time of a sample component, it is important
that every time a sample is analyzed,
standards or reference compound should be
run along with it. In the case of peak 2,
recorded retention time fall between the
retention times of stearic and oleic acid.
Based on the literature, stearic acid is not a
major fatty acid component of red bean but
in this study it yields high percent
concentration. This may be due to the
hydrogenation of unsaturated fatty acids
such as oleic, linoleic and linolenic acids
which leads to conversion to a saturated
fatty acid, which in this case, maybe stearic
acid.
IV. Summary and Conclusion
Figure 3. Chromatogram of fatty acid methyl
ester (FAME) component of red kidney bean
oil. Peaks from left to right: Hexane, Stearic
Acid ME (C18) and Oleic Acid ME (C18:1) or
Linoleic Acid ME (C18:2).
The physico-chemical attributes of food
products are important parameters to
consider because it dictates process designs
and manufacture of food products. Results
of this study show that red kidney bean has
low moisture content (12.31 ± 0.12), little
amount of ash (3.84 ± 0.28) and near to
neutral pH (6.7). The approximate, expected
C.J.Domingo / Journal of Food Composition and Analysis 1 (2014) 1−10
moisture content of a food can affect the
choice of the method of measurement. Ash
content represents the total mineral content
in foods. Determining the ash content is
important for it is a part of proximate
analysis for nutritional evaluation. The pH
value tells us that if the food product has a
pH less than 4.6, processing with boiling
water is enough to preserve the product
while those with pH 4.6 and above needs
processing under pressure. Total soluble
solids (2.73 °Brix ± 0.12) and color (L*
87.86, a* 0.17 and b* 12.82) can be used to
indicate quality of food products.
Red kidney bean oil gave positive result for
the presence of phospholipids. Results
showed the presence of phosphatidyl serine,
lysophosphatidyl ethanol amine and two
unknown phospholipids which were not
identified due to the limited reference Rf
values.
Gas chromatography is an indispensible tool
on the determination of fatty acid content in
different food samples. In this study,
identification of the fatty acids was based on
their retention time as compared to standards
and literature percent (%) concentration
values. Results indicated that the
chromatogram of red kidney bean oil had
shown 2 recognizable peaks and identified
as stearic acid ME (C18) and oleic acid ME
(C18:1) or linoleic acid ME (C18:2)
corresponding to concentrations of 16.50%
and 83.54%, respectively.
V. Recommendations
Although only possible phospholipids and
fatty acids present in red kidney bean oil
were reported, these claims have been based
in some theoretical aspects, other studies
and the experiment itself. Verification steps
are needed in order to really conclude that
these phospholipids identified from various
9
food samples are really the phospholipids
detected.
It is important to note that there are a lot of
factors that needs to be considered in order
to have a reliable, reproducible data after the
compositional analysis, thin-layer and gas
chromatography. Considerations will not
just for the sample preparation, but mostly
for the parameters that will be used or
applied in the analytical process.
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