Preparation of seed extracts and determining of protein and carbohydrate content

Abstract

The aim of the experiment was to determine the protein concentration and
test for the presence of carbohydrates in kidney beans and split peas seed
extracts. Seed extracts was prepared by soaking them in water overnight.
The kidney beans and split peas was grind fine and the liquid extract was
squeezed into a beaker through Miracloth. The liquid extract of the seeds
obtained were transferred into 15ml plastic tubes; centrifuged for 3minutes
at 3000rpm and refrigerated. Protein concentrations of seed extracts were
determined by the use of the Bradford method. The absorbance of 595nm
was recorded for both the diluted and true protein samples. The data of the
absorbance was used to construct a standard curve to determine the
concentration of the unknown protein sample. The kidney beans had a
concentration of (2,5mg /ml) and the split peas had a concentration of
( 8200mg/ml ). It was noticeable that the split peas are more highly protein
concentrated then the kidney beans. It was also observable that as the
dilution factor increases the absorbance decreased. Carbohydrates within
the seed extracts were tested with 2 required biochemical methods. Starch
was tested by the use of Lugol solution and benedicts reagents was used to
test for the presence of reducing sugars. The result was negative for testing
the presence starch in the seed extract and was observable as a milky
colour. The results for testing presence of reducing sugars using the Bradford
method with the seed extract of kidney beans was positive, indicated by a
green colour and white precipitates were noticeable within seed extract of
split peas. Thus it is evident that split peas has sugars present, but in limited
amounts. No starch was present in the seed extracts; however there were
signs of reducing sugar present. It is then evident to conclude, that split peas
has a higher concentration of proteins than kidney beans, but kidney beans
on the other hand has more reducing sugars present.
Introduction

The objective of the study was to determine the protein concentration and
test for the presence of carbohydrates in kidney beans and split peas. There
are different types of methods for determining the concentration of protein
like the Lowry assay and BCA assay. The protein concentration was
determined by the use of the Bradford method. The Bradford method uses
dye, which has a negative charge that binds to the positive charge of the
protein (Abrahams et al., 2010). The more protein concentrated the seed
extracts of both kidney beans and the split peas were, the more dye have
bind, which made the colour more darker. Therefore the test tubes ,1-5
ranged from dark blue to light blue, indicating that the protein concentration
have become less after the dilution of the seed extracts were made. Bovine
serum albumin (BSA) with known concentration and Bradford reagent was
used to find the absorbance at 595nm for each of the 6 seed extracts and
the diluted seed extract samples. This was used to construct a standard
curve to determine the unknown concentration of the protein sample of the
seed extracts. Proteins are essential /biological important to all organisms
because of its many diverse functions like support, enzymes, transport,
defense, hormones and motion. Most cells have 50% dry weight that consists
only out of proteins (Mader, 2007). Therefore proteins are a useful source to
humans and other organisms. Carbohydrates are also an energy riched
source and are basically sugars and starch. Organisms store carbohydrates
in the form of polysaccharides rather than monosaccharide’s.
Monosaccharides are simple sugars, like glucose and fructose. Disaccharides
would be two simple sugars attached, like sucrose and polysaccharides
would be a long chain of monosaccharides. Starch is produced by plants and
stored as energy. Starch is found in rice, potatoes, fruit, seeds and beans.
Carbohydrates were determined in seed extracts with 2 biochemical test.
Starch was tested using Lugol solution and sugars were tested with the
required Benedicts reagent (Benedict’s tests). Testing starch with Lugol
solution changes colour from yellowish-brown to dark blue. No colour
changes would be observed if monosaccharide or disaccharides is present in
the seed extracts. Benedict’s reagent was used in testing reducing sugars.
Carbohydrates with free aldehydes and ketones group have reducing
properties in alkaline solutions. The reducing ends will react with the
Benedicts reagent forming a coloured precipitate (Abrahams et al., 2010). A
positive result testing for starch would be indicated with a dark blue colour
and a positive result for testing for the presence of reducing sugars would be
indicated in a red, green or yellow colour. Split peas and kidney beans
indicated a milky /cream colour for the presence of starch, thus it is a
negative result for starch. Reducing sugars in kidney beans was indicated
with a green colour. Split peas had white precipitates indicating that there
are reducing sugars present but in limited amounts. Understanding the
useful sources derived from seed extracts like protein ,and carbohydrates ,it
will enable us to know what seed extract between kidney beans and split
peas is more energy rich sources(Rehman et al., 2001).Therefore the
information obtained from the study can be beneficial and important to
humans and other animals .

Materials and methods

1) Preparation of seed extracts

A 10g of each seed type (kidney beans and split peas) was weighed with a
required scale. The seeds were then place in a 50ml plastic tube and water
was added to soak each seed type. This method was done to ensure that the
different seed types extract water and get softened at the same time. The
following day the excess water was pour off from the soaked seeds with in
the 50ml plastic tube. The seeds were then placed into a mortar and 10g of
white quartz sand was added. The mixture was then ground within the
mortar using a pestle. A 10 ml of 0.1M potassium phosphate (ph 7) buffer
was added. The 0.1M potassium phosphate (ph 7) buffer mix was prepared
as follows:

Mass= Mwt*C*V

Therefore 0.136g of potassium phosphate was added in a 50ml plastic tube

with the addition of 8ml distilled water. The ph of the mixture was then
buffered to a ph 7 and the remaining 2ml of this was added to a final volume
of 10ml. the mixture was grinded further for another 5minutes with using a
pestle. A 10ml of 0.1M potassium phosphate (ph 7) was added again and mix
with a required spatula. The grind mixture was then poured in a Miracloth
and the liquid extracted was squeezed into two 15 ml plastic tubes and to
ensure that the squeezed mixture was equally distributed within the two
15ml plastic tubes. The two test tubes was labeled and centrifuged for
3minutes at 3000rpm. After the two 15ml plastic test tubes were centrifuge,
the upper liquid phase was then transferred into another two clean 15ml
plastic test tubes that was labeled respectively and refrigerated till when
needed.

2) Determining protein concentration

The performed experiment, two separate solutions, Bonvine serum albumin

(BSA) and Bradford reagent was supplied by the practical co-coordinator .

PROTOCOL FOR THE BRADFORD ASSAY.

REAGENTS TUBE NUMBER

1 2 3 4 5 6
0.1mg/ml 0 0.2 0.4 0.6 0.8 1
BSA
WATER 1 0.8 0.6 0.4 0.2 0
(ml)
BRADFOR 5 5 5 5 5 5
D
REAGENT(
ml)
TOTAL 6 6 6 6 6 6
VOLUME 2.1)
Preparation of BSA concentrations for standard curve.

Six clean 15ml plastic tubes were labeled ranging from 1-5 and used to
prepare six different concentrations of BSA solution .in each test tube a
different volume of stock solution of a concentration of 0,1mg/ml and a
different volume of water was added .thereafter 5ml of Bradford reagent was
added to the solutions in each tube the final concentration of these
solutions were then calculated using C1V1=C2V2 and the values were
plotted to form a protein standard curve graph. (CALCULATIONS OF
CONCENTRATIONS TO CONSTRUCT THE GRAPH WOULD BE AT THE END OF
THE METHOD AND MATERIALS)
2.2) Serial dilution for split peas and kidney beans

Firstly two 15ml tubes ,one containing split peas and another containing
kidney beans was available from a previous experiment .three clean glass
test tubes were then labeled [ , , ] for kidney beans and three[
, , ] for split peas .in the first set of test tubes 1ml of kidney
beans extract an 9ml of water was added to test tube 1. Thereafter 1ml of
test tube 1 solution +9ml of water was added to test tube 2. Finally 1ml of
test tube 2 solution and 9ml of water was added to test tube 3.the same
procedure was followed for the split peas extract in the remaining three test
tubes . followed by this step , 1ml of each of the dilutent was extracted and
place in six 15ml plastic tubes labeled kidney beans[ , , ] and
split peas[ , , ] then 5ml Bradford reagent was added to all the
tubes .

2.3) Absorbance measured in (nm)

A spectrophometer was used to measure the absorbance of the different

diluted solution concentrations the solutions containing stock solutions of
BSA (0,1mg/m) previously mentioned was used for this part of the
experiment. an Aliquot amount of each solution from tube 1-6 was placed in
the spectrophotometer .the spectrophotometer was blanked at an
absorbance of 595nm when starting with tube 1 thereafter the absorbance
for the remaining ( 2-6 ) tubes were measured and results were recorded in
a table.

3) Determining the carbohydrate present in each of the seed extracts

(kidney beans and split peas)

Seed extracts was 1st tested for the presence of starch. A 2ml seed extract
solution (kidney beans and split peas) into one clean 15ml plastic tube and
add 2ml of distilled water to another clean 15ml plastic test tube. To each of
the test tubes 3-4 drops of Lugol solution was added. The two 15ml plastic
test tubes were then gently swirl to allow mixture to mix and left for colour
change. The colour change was then observed and recorded. Reducing
sugars that were tested for each of the seed extracts (kidney beans and split
peas) with the required Benedicts test. A 1ml for each of the seed extracts
was added to one glass test tube that was labeled with the seed extract
(kidney beans or split peas) that was use and another 1ml of distilled water
was added to another glass test tube and labeled as the control. To each of
the glass test tubes a 5ml of Benedict’s reagent was added and the two glass
test tubes were then heated in hot boiling water for approximately 5minutes.
After the 5 minutes the two glass test tubes were then checked and
observed for any colour change and the results was then recorded.

CALCULATIONS FOR PROTEIN STANDARD CURVE:

METHODS USED FOR SERIAL DILUTIONS:

RESULTS:

MEASUREMENTS OF ABSORBANCE AND DILUTED ABSORBANCE FOR EACH

SEED EXTRACT SOLUTION, IN THE SIX TEST TUBES:
TABLE 1:

GRAPH 1(THE STANDARD CURVE AND OBSERVED RECORDED ABSORBANCE

VALUE GRAPH IS CONSTRUCTED ON GRAPH PAPER AND INDICATED AS
GRAPH 1)

TUBE NO ABSORBANCE VALUE OBSERVED

KIDNEY BEANS SPLIT PEASE
1 0 0
2 0.296 0.354
3 0.406 0.512
4 0.507 0.718
5 0.506 1.387
6 0.578 1.110
DILUTED KIDNEY TEST TUBE :1 1.021
BEANS SEED EXTRACT TEST TUBE :2 0.433
SOLUTION(ABSORBAN TEST TUBE :3 0.366
CE)
DILUTED SPLIT PEAS TEST TUBE :1 1.627
SEED EXTRACT TEST TUBE :2 1.137
SOLUTION(ABSORBAN TEST TUBE :3 1.017
CE)
CALCULATION FOR DETERMINING THE PROTEIN CONCENTRATION FOR EACH
SEED EXTRACT

TABLE 2: INDICATORS FOR TESTING THE PRESENCE OF STARCH AND

REDUCING SUGARS IN EACH SEED EXTRACTS.

INDICATION OF STARCH PRESENT CODE

NO STARCH 0-
TRACE OF STARCH +-
LITTLE/SOME STARCH ++-
MUCH STARCH +++-

INDICATION OF REDUCING SUGARS CODE

PRESENT
NO SUGAR 0-
YELLOW +-
GREEN ++-
RED +++-

TABLE 2.1

AMOUNT OF STARCH OBSERVED

SAMPLE TESTED CODE

WATER 0-
KIDNEY BEANS 0-
SPLIT PEAS 0-

TABLE 2.2

AMOUNT OF REDUCING SUGARS OBSERVED

SAMPLE TESTED CODE

WATER 0-
KIDNEY BEANS ++-
SPLIT PEAS 0-

Discussion

It was noticeable that the experiment done to determine the protein in the
seed extracts had different protein concentrations. The was observed that as
the BSA concentrations increased in the six different test tubes, the colour of
the solution went from light blue to dark blue. This was due to the Bradford
assay principle. The reaction occurred when the negative charge of the dye
bind to the positive charge of the protein (Abrahams et al., 2010). The serial
of dilutions had indicated another observation. It was found that as the
concentration decrease for the diluted extracts, the colour change of the
solution went from dark blue to light blue. This was due to the fact that test
tube 1 had a higher protein concentration than the rest of the diluted test
tubes. When the absorbance was measured by the spectrophotometer, the
same comparison was obtained. The test tube for the highest absorbance
was found in test tube 1, whereas the rest of the diluted test tube had lesser
absorbances. The absorbance increased the proteins concentration
increased. Split peas had a higher absorbance of 1.017 and kidney beans
had a lower absorbance value of 0.433 as shown in table 1. The absorbance
increased as the BSA concentration increase thus producing a standard
straight curve. It was clearly noticeable in graph 1 that split peas were more
protein concentrated then kidney beans. Split peas had a protein
concentration of (00), whereas kidney beans had a protein concentration
of(00). Thus it is evident to state according the results, split peas is more
protein concentrated and useful in the sense of getting proteins
consumption. It was noticeable in table 2.1 with testing the split peas and
kidney beans for starch using Lugol solution, that a milky, cream colour was
observed in both cases of the seed extracts. Therefore it is suggested that
starch test negative (no starch present) for split peas as well as kidney
beans. The water served as a control throughout the experiment and would
not react nor indicated a colour change (result). Results obtained in table
2.2, testing both split peas and kidney beans for reducing sugars using
Benedicts reagent suggested as followed. Kidney beans had a positive result.
The colour changed observed for kidney beans seed extract was green (+
+-). It was noticeable that white precipitates were found within the split peas
sample. Split peas had no colour change implying a negative result (0 - no
sugars). Therefore a clear feature that indicates hat split peas have limited
or no reducing sugars. It is evident that there are no starch present for each
of the seed extracts (kidney beans and split peas), but kidney beans had
reducing sugars present and split peas had none
CONCLUSION

The aim of the experiment was achieved. The results suggest that that there
are no starch present for each of the seed extracts (kidney beans and split
peas), but kidney beans had reducing sugars present and split peas had
none. Sources derived from seed extracts like protein and carbohydrates will
enable help in knowing what seed extract between kidney beans and split
peas is more energy rich sources. Metabolical functions like respiration,
where glucose (reducing sugar) is broken down for energy (A.T.P.) relies on
these energy rich sources. Cells in the body depends on this useful nutrients
derived like carbohydrates and proteins for other diverse function as well. It
is also evident that as the dilution factor increases, the absorbance
decreased. Therefore split peas is more protein concentrated then kidney
beans.(results)

Reference:

• Abrahams, Z and Nene, S.(2010) ‘Biomolecules &cellular Processes’

BTN 216 PRACTICAL MANUAL,University of the Western Cape, Faculty
of science, Department of Biotechnology

• Eyaru, R., Shrestha, A. and Arcot, J.(2009) ‘Effect of various processing

techniques on digestibility of starch in Red kidney bean (Phaseolus
vulgaris) and two varieties of peas (Pisum sativum). Elsevier, 42 (2009)
956–962(online cited) (9 March 11, 2010) AVALABLE FROM:
http://www.linkinghub.elsevier.com/retrieve/pii.com

• Mader, S. (2007)’CELL STRUCTURE AND FUNCTION’, 9th edition,

Biology, New York, McGraw-Hill Inc. Pg 82.
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available carbohydrate content and starch digestibility of kidney beans
( Phaseolus vulgaris L.)’ Food chemistry, personnel review, 73(2001),
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