ELECTROPHORETIC AND SPECTROSCOPIC CHARACTERISATION OF

PROTEIN ISOLATE IN DELONIX REGIA

BY

ADEDAYO, Suliyat Motunrayo

H/ST/20/1817

DEPARTMENT OF SCIENCE LABORATORY TECHNOLOGY,

FEDERAL POLYTECNIC ILARO
OGUN STATE

JUNE, 2022
 ELECTROPHORETIC AND SPECTROSCOPIC CHARACTERISATION OF
PROTEIN ISOLATE IN DELONIX REGIA

BY

ADEDAYO, Suliyat Motunrayo

H/ST/20/1817

PROJECT SUBMITTED TO THE SCHOOL OF PURE AND APPLIED

SCIENCES,
FEDERAL POLYTECHNIC ILARO

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

THE HIGHER NATIONAL DIPLOMA IN SCIENCE LABORATORY
TECHNOLOGY (CHEMISTRY)

November, 2022

i
 CERTIFICATION

This thesis titled: ELECTROPHORETIC AND SPECTROSCOPIC

CHARACTERISATION OF PROTEIN ISOLATE IN DELONIX REGIA writing

by ADEDAYO, Suliyat Motunrayo (H/ST/20/1817), meets the regulations governing

the award of Higher National Diploma of Federal Polytechnic Ilaro and it is approved

for its contribution to scientific knowledge and literary presentation.

Dr. Oyedeji, A. O.
___________________
SUPERVISOR
SIGNATURE & DATE

Mr. Afolayan, A. O.
___________________
Head of Department, (SLT)
SIGNATURE & DATE

ii
 DEDICATION

This project is dedicated to Almighty God, whom we can give thanks for successful

completion of this program.

iii
 ACKNOWLEDGEMENTS

The administrative and academic influence of the Head of the department of Science

Laboratory Technology was duly acknowledged in the person of Mr. A. O. Afolayan, I

am most grateful for your orientation and profound technical insight into science

aspects of this work.

To my supervisor Dr. A. O. Oyedeji who has especially been pivotal to the success of

this work, I thank you immensely for reading through all scripts, for your patience and

expertise advice and for the many constructive criticisms that have greatly enlightened

us.

My Special thanks go to the entire staff of the department of Science Laboratory

Technology for the knowledge they have imparted in us within the classroom, field and

outside for the success of this project.

To my dearest parents, I say ‘a very big thank you, for your support both financially and

morally at this level of my life. I pray they will live long to eat the fruit of labour. It is a

must for me to express my gratitude to Mrs Oyebanji and my beloved uncle Mr

Adedayo Mudashir, Mr Adedayo Rasheed and Mr Fatai Kehinde and to my dearest

brother Oyebanji Olayinka and Oyebanji Mustapha and my dearest sister Oyebanji

Abidemi, Hassan Abisoye and Ajayi Bukola and to the entire family of Adedayo for

their support throughout these project, they will all live long on earth.

Finally, to my project mates, I really appreciate your support, cooperation, hard work

during the project, and to all I could not mention their names, may God bless you all

and may will all succeed in life.

To friends and family, thank you all immensely for your priceless pieces of
iv
encouragement; am truly grateful.

TABLE OF CONTENTS

Contents
Page

CERTIFICATION..........................................................................................................ii

DEDICATION................................................................................................................iii

ACKNOWLEDGEMENTS...........................................................................................iv

TABLE OF CONTENTS................................................................................................v

LIST OF TABLES..........................................................................................................vi

LIST OF FIGURES......................................................................................................vii

LIST OF PLATES........................................................................................................viii

ABSTRACT....................................................................................................................ix

CHAPTER ONE..............................................................................................................1

1.0 INTRODUCTION.........................................................................................1

1.1 Background of the Study.....................................................................................1

1.2 Statement of the Problem....................................................................................3

1.3 Justification of the Study....................................................................................4

1.4.1 Aim of the Study...............................................................................................4

1.4.2 Objectives of the Study....................................................................................4

CHAPTER TWO.............................................................................................................5

2.0 LITERATURE REVIEW..............................................................................5

CHAPTER THREE……………………………………………………………………………………
6

3.0 MATERIAL AND

METHOD……………………………………………………………….7

4.0 RESULT AND

v
DISCUSSION……………………………………………………………...8

5.0 CONCLUSION AND RECOMMENDATION………………………………………………9

REFERENCES................................................................................................................5

LIST OF TABLES

Table

Page

1.1 Nutritional Status with Body Mass Index

42

vi
 LIST OF FIGURES

Figure

Page

2.1:

vii
 LIST OF PLATES

Plate

Page

viii
 ABSTRACT

Body mass index diagnostic machine can be improved upon by including special
additional features such as image capturing and data logging sub-systems, as these
would solve the problems of improper data documentation/referencing, user status
monitoring and data incoherence. This study design and construct automatic body mass
index framework. The material for the design and construction are high density fiber
(HDF) and acrylic casing. The system is powered with 2A/5V adaptor, and the 8 Mega
pixel camera stand is adjustable to cater for various heights. The output live image of
the user is displayed on 7inches raspberry pi smart screen of 1024 x 600 resolution
capacity on GUI which contains a capture button that takes the user image and stores it
for future call. Python programming language is used to instruct the raspberry pi for
necessary in instructions in controlling the sensors and to design the system GUI. The
system was calibrated, tested and analyzed, and found to be efficient in terms of
execution time, visual display quality, data retention and recall. It is recommended that
these additional features should be included as modifications and improvement to
modern BMI machine to solved the existing problems.

ix
x
 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the study

Because animal protein is out of reach for a large number of individuals in underdeveloped

nations, legumes are utilized to reduce the problem of protein malnutrition in Africa (Lawal et

al., 2005). Inadequate supplies and a lack of dietary protein have necessitated the search for

novel legumes as a new source of protein for use as a functional food ingredient and nutrition

supplement (Shahidi et al., 2001). Most recent studies have focused on the use of plant protein

in the creation of novel products or conventional foods. Seed protein offers vital amino acids,

but they must also have the necessary functional characteristics to be used in a variety of

formulations. Protein functional characteristics are responsible for many of the factors that

influence consumer acceptability of food items; as a result, they play an important role in food

processing and product creation (Boye, et al., 2010). When consumed, legumes are the edible

fruits or seeds of plants that give protein. They are members of the Leguminosae family and are

widely grown around the world (Ayodele & Ade-Omowaye, 2015).

Protein extraction is a frequent procedure in biological research. For preparation of

plant cell extracts, it is usually required to grind and homogenize plant materials to

physically break the robust cell wall. Sample grinding is laborious and time-

consuming, especially when a large number of samples are handled at once. In the

case of yeast cells, which also have a cell wall, proteins can be efficiently extracted

after cells are treated with alkaline (NaOH) and boiled in SDS-containing solution

(Kushnirov, 2000).

A lot of trials had been made on the use of plant proteins including soya bean,

groundnut and cotton seed cakes. They are however, deficient in essential amino

acids (Oyegbile, Yola and Abdullahi, 2017). Plants protein consumption by

xi
livestock and humans has led to their scarcity and high cost therefore, there is need

to search for alternative feedstuff which will be available and cost effective. The

necessity of producing protein rich foods from plant protein to augment animal

proteins is to contribute positively not only a nutritious diet but also to food security

and sustainability (Claudine et al., 2020). To compensate for the future paucity of

animal proteins, the industry and consumers are focusing their attention on plant

protein. Plant protein isolates are often used to create food because of their

functional properties, protein content, sustainable production, and relatively low

cost (Audrey et al 2019). Not only as a micronutrient, proteins play essential roles

in food but also as structural ingredients which aid in the formation of food gel and

contribute to the desired texture and mouth feel attributes of the final food product.

However, with the continuous growth in the world population the demand for

protein for human consumption is increasing and a number of alternative source are

being explored to meet this demand. Among them plant proteins have received

particular attention mainly on the ground of enhanced environmental sustainability.

The acceptance of plant proteins by consumers is also greater than that of animal

proteins and the blend of several protein sources is usually required to ensure

sufficient amounts of essential amino acid (Laura and Samantha 2021). The amino

acid profile of plant proteins is less favourable than that of animal proteins, mixing

protein from different sources may not only result in a more balanced amino acid

up take but also in techno functional synergies. From a nutrition point of view,

legumins are of particular interest due to their higher content of sulfur containing

amino acids (S-AA) compared to other globulins. Therefore, cultivars of legumes

with higher proportions of sulfur-rich proteins will be of great interest in human

nutrition. On the other hand, protein properties such as solubility, heat-induced

xii
gelation, foaming and emulsifying capacity are essential for the food processing

industry. (Ahmed, Nicholas, Donal and Paola 2022).

Delonix regia (Flame of Forest)

Delonix regia is a shallow-rooted, prominent, fast-growing, virtually evergreen

legume tree that can reach a height of 10-30 m. In locations with a long dry season,

trees might drop their leaves. The bole can be quite short at times. The trunk can

grow up to 2 meters in diameter and be buttressed at the base. The bark is smooth,

with lenticels, and is sometimes slightly cracked. The crown is umbrella-shaped,

with long horizontal branches that spread widely. As a result, the total diameter of

the tree is greater than its height. When young, the twigs are robust, greenish, and

finely hairy before turning brown. The leaves are bipinnate, alternate, feathery, 20-

60 cm long, with 10-25 pairs of pinnae, each with 30-60 opposing leaflets, and are

bipinnate, alternate, feathery. The leaflets are 0.5-1 cm long, stalkless, and hairy on

each side The inflorescences are mildly scented corymbs that are borne laterally at

the twig ends. Large (5-13 cm), gorgeous orange-red blooms are freely grouped on

5-7.5 cm long stalks in the inflorescence. The four clawed petals are spoon-shaped,

while the five sepals are thick, green, and coarsely hairy. The fruit is a 30-75 cm

long pod that starts off green and flaccid, then turns brown and woody as it matures.

After the tree has shed its leaves, the pods remain on the tree. The 30-45 seeds in

each pod are firm, greyish, speckled, and oblong in shape, like date pits. They have

a hard coating on them. Delonix regia is named after the Greek words "Delos"

which means "visible" and "onyx" which means "claw." (Rojas-Sandoval et al.,

2013; Orwa et al., 2009)

xiii
Common names of D. regia are flamboyant, royal poinciana, flame tree, fire tree,

flame of the forests, gold mohar, peacock flower, poinciana, red tree, royal

gulmohur; royal peacock [English]; Flamboyant [French]; Feuerbaum,

Flammenbaum [German]; flamboaiã,acácia-rubra, uaruna [Portuguese]; acacia

roja, acacia rubra, arbol del fuego, chivato, , clavelino, flamboyant colorado, ,

framboyán; rojo, flor de fuego, flor de pavo, guacamaya, josefina, malinche,

morazán, poinciana, tabuchín [Spanish], Phượng vĩ [Vietnamese]; ‫رنف ملكي‬

[Arabic]; mjohoro,mkakaya [Swahili]; sekeseke [Yoruba]. (Rojas-Sandoval et al.,

2013; Orwa et al., 2009)

Uses of Delonix Regia

The flamboyant's primary function is ornamental. It is commonly used as a shade

tree for cows or other tree species in plantations, such as tea plantations, and is

commonly planted in parks and avenues for its wonderful flowers. However,

because the tree has been known to fall over unexpectedly, it is no longer as

popular as a street tree in South Africa (Roux, 2003). Delonix regia seed meal can

be fed to farm animals, while the leaves provide feed for livestock. Bees find the

blooms to be a nice and plentiful source of food. Firewood can be made from wood

and woody pods. The wood can be used for light construction, fence posts, and

pirogues, among other things. The seeds can be used in collars as pearls. as a source

of heat. The wood can be used for light construction, fence posts, and pirogues,

among other things. The seeds can be used in collars as pearls. The leaves and

flowers have been claimed to have herbicide effects on Mikania micrantha, an

invasive climber, and the bark is used in ethnomedicine. Delonix regia wood ash

has the potential to drastically eliminate fungus and insects (Rojas-Sandoval et al.,

2013; Orwa et al., 2009).

xiv
1.2 Statement of the problem

Because animal protein is in short supply due to population growth, we must

supplement our protein supply with underutilized plant seeds. Shortage of animal

protein has resulted in severe malnutrition in growing children and women of child

bearing age.

1.3 Justification of the study

To ameliorate protein shortage resulting in malnutrition, it is necessary to explore

an alternative plant protein because of its availability and renewability.

1.4 Aim and Objectives of the Study

1.4.1 Aim of the Study

The aim of the study is to isolate and characterize whole protein of D. regia

1.4.2 Objectives of the study

The objectives of this study are to:

i. isolate the protein of defatted D. regia seed using the alkaline precipitation

method;

ii. dialysis of the protein isolate;

iii. freeze-dry the sample to lyophilize it

iv. perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) on the protein isolate;

v. FTIR analysis to predict the functional group and amino acid type

vi. Protein content in the sample using spectroscopic method

vii. Kledjahl method analysis (digestion,distillation,titration)

xv
xvi
 CHAPTER TWO

2.0 LITERATURE REVIEW

Extraction, gelation and microstructure of Bambara groundnut (BGN) vicilin was

carried out by Claudine, Linda, Victoria, Paul and Erik (2019). Vicilin was extracted

from defatted BGN flour using an optimized Osborne fractionation method. The protein

content was measured with the Dumas nitrogen combustion method, the native PAGE

analysis was performed to establish the electrophoretic profile of BGN vicilin proteins

in their native state, differential scanning calorimeter was used to determine the thermal

properties of BGN vicilin solutions. Vicilin protein gels at three protein concentration

(4.4, 9.2 and 12.7%) in the presence of 200 mM NaCl was prepared for SEM analysis.

Protein content of BGN vicilin was found to be 91.4±1.0 g/100 g, with minor amounts

of fat, moisture, ash and carbohydrates being 0.4, 0.2, 2.2, and 5.8% respectively. The

native-PAGE profile of BGN vicilin and BGN flour gave two major bands at

approximately 170 and 385 kDa, respectively. The denaturation temperature of BGN

vicilin was observed at 92 and 94 °C with respect to absence and presence of NaCl.

From the CLMS images, no structural information was distinguished for the protein

concentration range investigated which indicates a homogeneous system. From the

image, a Df close to 3 was assumed. From SEM images structures were observed in a

range length scales from 3 µm down to submicron lengths.

Physicochemical and functional properties of lentil protein isolates prepared by different

1
drying methods was carried out by Joshi, Adhikari, Aldred, Panozzo and Kasapis

(2011). Deshelled lentil was dispersed in distilled water and the pH was adjusted to 8.0

with 1N NaOH, protein sample was first frozen for 24h at 18 ° C for freeze drying,

Thermal characteristics of all lentil protein isolates samples were analysed using a

differential scanning calorimeter, Textile profile analysis of the lentil protein isolate gel

samples was performed using a TA.XT plus analyser to determine their mechanical

properties, lenticel protein samples were analysed using the standard methods for

moisture, crude protein and ash content. Lentil protein isolate samples colour was

measured using a chroma-meter, surface morphology of protein isolate powders was

studies by viewing under a scanning electron microscope, the protein profiles of three

LPI samples was carried out to investigate sodium dodecyl sulphate –polyacrylamide

gel electrophoresis(SDS-PAGE). Rheological properties were measured with rheometer

and the changes in rheological properties of protein dispersion during thermal treatment

was studied by monitoring storage and loss modulus (G’’ and G”) and loss tangent of

the gels. Lenticel protein isolate (LPI) powders prepared by all the three drying method

the chemical composition (protein and ash contents). Protein and ash contents of the

dried isolates samples varied from 90.21 toB91.85% g protein/100 g dry protein isolate

and 3.19 to 3.83%(dwb) respectively. Micrographs of lentil protein isolate powders of

the scanning electron microscope reveal that spray dried protein isolate appears to be

more uniform in particle size distribution compared to the freeze- dried and vacuum-

dried protein isolate, through laser diffraction the particle size distribution data of freeze

dried and vacuum dried with a very large size were obtained. The SDS-PAGE results

2
show that lentil protein has a complex protein profile with more than 20 protein bands

with 23 major bands in the ranges of 40-85kDa. The stress relaxation behavior of the

three LPI gels shows a gradual decrease in stress over time and attained equilibrium

stress and the gel formation behavior of heat induced lentil protein isolate gels was

studied by monitoring storage loss modulus.

According to Cosson, Olivera, Descamps, Saint-Eve and Souchon 2022) characterized

the peptides in pea protein isolates using high performance liquid chromatography with

mass spectrometry and bioinformatics tools. Different pea protein fractions 3,005

unique peptides representing various protein families, some of them were correlated to

having broth and bitter attributes particularly, short sequences (<8) residues. Upon the

preliminary examination of the peptides, 3561 peptide ions and 3005 unique peptides

were identified respectively. The peptide had origin from storage protein, enzymes and

protein from seed metabolism. The peptides were mostly polar and hydrophilic. With

respect to amino acid content, the peptides were aliphatic amino acids (Ala, Val, Leu,

and Ile) the aromatic amino acid (Phe, and Tyr) or acidic amino acid group (Asp, and

Glu )

According to Danno, Kanazawa and Natake (1974) extracted wheat flour protein with

sodium dodecyl sulphate and their molecular weight distribution. Sodium dodecyl

sulphate at various concentration and to extent protein from wheat flour of PH 6.8. At

least 14 bands were observed although two very densitometry trace. The most intense

bands (9-12) in no reduced SDS-soluble protein correspond to a polypeptide of

molecular weight 30,000-35,000. The faint, broad brand (band1) were observed in
3
higher molecular weight region and trace amounts of materials did not enter to the gel

during electrophoresis. The best reagent for extraction of wheat flour nitrogen and about

76%.

Faba bean protein (FBP) films reinforced with cellulose nanocrystals as edible food

packaging material was studied by Sandra et al. (2021). The FBP was prepared by

solution casting process, it was done by dissolving FBP (5% w/v), 50% glycerol based

on the dry weight of FBP) and distill water. The thickness of the FBP was known at ten

random points around each film sample using a manual micrometer, the mechanical

properties, tensile strength, the young’s modulus and the elongation at break of FBP

were determined. Different thermal behavior of FBP films acquired were assayed by

thermogravimetric analysis using a Mettle-Toledo TGA/DSC 1 thermobalance. By

measuring the weight loss of films, Moisture Content of FBP films samples was

determined gravimetrically.

Structural characterization of protein isolates obtained from chia (Salvia hispanica L.)

seeds was studied by Debora et al. (2017). The structural properties of characterization

of protein isolate CPI obtained, the effect of mucilage removal, the alkali pH for protein

extraction (8,10,12) and the acidic pH used for the isoelectric precipitation (3or 4.5)

were evaluated. with some modification protein isolation was performed according to

Timilsena et al. (2016), samples were centrifuged for 15min at 10000g in order to

remove the mucilage, for protein extraction, the pH OF the slurry was adjusted to 8,10

or 12with 1mol/L NaOH and stirred for 1h. This ensure the solubilization of the

maximum amount of protein extracted and samples were freeze-dried. The protein
4
content of CPI was determining by using kjeldahl procedure(AOAC,1970), using a

conversion factor of 6.25, According to Laemmli method (1970) by using sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine

electrophoretic pattern of both CPI, In order to determine both thermogravimetric (TG)

and derivative thermogravimetric (DTG) curves samples of CPI were heated from room

temperature to 800ºĊ, Fourier transformed infrared spectroscopy was use to study the

structure and stability of protein in a wide variety of environments, infrared spectra

were measured at room temperature in a Shimadzu IR-Affinity-infrared

spectrophotometer (Shimadzu co.., Duisburg, Germany).The freeze-dried powder was

analyzed by using GladiATR monolithic diamond crystal accessory(Pike Technology,

Madison<USA).In Differential scanning calorimeter an aliquots (10-15mg)of

dispersions (200g/kg in distill water )was sealed in coated standard aluminum pans. The

electrophoretic pattern of both CPI were determined by SDS-PAGE under reducing

condition both samples showed a similar protein profile with a large number of protein

bands, the TG and DTG plots reveal that three stages take place during the pyrolysis

process of CPI, Slight changes in wavelength of protein isolate may result from

differences in functional group, amino acid composition and interaction among them.

Seed Development and protein Accumulation Patterns in Faba Bean (Vicia faba,L.)was

carried out by Ahmed,Nicholas,Donal,Paola (2022).De pace et al used one-dimensional

sodium dodecyl sulfate –polyacrylamide gel electrophoresis (ID SDS-PAGE) to

investigate the accumulation of few vicilin and legumin band while on the other hand,

Panitz et al studied vicilin and legumin accumulation by using a single cDNA ,Crude

5
Protein Content Analysis seed were freeze-dried until a constant weight was obtain

depending on the sample availability 60 to 100mg of seed flour was analyzed in a

LECO carbon, hydrogen, nitrogen content was converted to protein content using the

5.4conversion factor, Total Protein Extraction was conducted as described by Scollo et

al to remove phenolic compounds a cold (̴ 4ºĊ) aqueous acetone (80℅v/v) sodium

ascorbate containing 5mM was added to the samples at a 1:20 sample to buffer ratio, 1D

SDS-PAGE Protein Profile of the Seed Developmental Stages. 1-D SDS-PAGE seed

protein profile shows a clear timeline of the accumulation of different seed proteins

across the 12growth stages.

According to Luis, Adriana, Santiago, Gloria and Maria (2007) Carried out

Physicochemical and Structural Characterization of Lima Bean (Phaseolus lunatus)

globulins, Characterization of P.lunatus globulins the protein were subjected to the

following tests;

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), the procedure

described by Martinez ,Castellani and Anon(1997) the following protein molecular mass

standard were use phosphorylase b, bovine serum albumin, overlbumin,carbonic

anhydrase,trypsin inhibitors,lactalbumin (94,67,45,30,20.1,14.4 kDA) respectively ,

Differential Scanning Calorimetry (DSC) according to the method of Martinez and

Anon(1996)protein fraction were analyzed by DSC samples were prepared in distilled

water (final concentration 25℅w/w),aluminum hermetic (Perkin-Elmer No. 0219-0062)

were loaded with 8mg of the 250g dispersions and allowed to stabilize for at least

30min at room temperature(25ºĊ) before testing, Globulin was analyzed by non-

6
denaturing and denaturing electrophoresis ,under non-denaturing conditions it presented

two bands the one of higher mobility more wide and intense than the one of lower

mobility. When analyzed under denaturing condition the bands of higher intensity

corresponded to polypeptides of 114-110kDa, 54±1kDa and 16±1kDa while less intense

bands corresponded to polyketide of 43.8±0.6kDa, 40±1kDa, 34±1kDa, 31.2 ±0.8kDa,

26.0±0.8 kDa, 23.0±0.5kDa respectively.

According to Mohammed et al. (2017) studied the fractionation, Physicochemical and

Structural Characterization of Winged Bean Seed Protein Fractions with Reference to

Soybean. Extraction of water, NaCl, NaOH and ethanol soluble proteins, winged bean

and soybean seed were manually cleaned, dehulled, ground using variable speed rotor

and passed through sieve to yield particle size flour. Colour... protein sample contained

a transparent glass cuvette uniformly size (0.5mm) was place in a calorimeter with black

and white tiles equipped with a tri-stimulus. Sodium dodecyl sulphate polyacrylamide

gel electrophoresis of protein fractions. Molecular size was determined by sodium

dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE) according to

Laemmli. The SDS-PAGE was carried out on a mini Precast Gel slab 10 wells in an

SDS-Tris-glycine buffer system, protein was prepared for freeze drying in non-reducing

buffer solution and heated at 95ºĊ for 50s, Protein Content of the Fraction was

determined by an automatic Kjeldahl unit according to the manufacturer instruction

following method 976.05 of the AOAC yield of each was calculated based on the

fractions obtained, By using a Differential Scanning Calorimetry the thermal properties

of proteins was determined a protein samples suspended in 0.01 m phosphate buffer (pH

7
7.0) were placed in aluminum pans equilibrated to 24ºĊ and scanned from 25 to 180ºĊ

at 10ºĊ/min. FTIR Spectroscopy analysis using FTIR spectrophotometer spectral data of

the proteins were collected (SPECTRUM-100, Perkin Elmer) fitted with universal

Attenuated Total Reflectance ,bands spectra were acquired within the mid IR range

(4000-800 cm) at a resolution of 2cm with 32 scans and corrected for background

absorbance by subtraction of the empty ATR crystal spectrum. The average protein

content of the raw winged bean seed was 38.31℅ and increased to 51.80℅ after hexane

extraction of the lipids, the winged bean albumin, globulin and glutelin fractions had

their pH at 4.0, 3.5 and 5.0 whereas the corresponding soybean fractions had pH at 3.6,

3.5, and 5.0 respectively, The colour of the winged bean and soybean protein value

varied significantly from 3.00 to 3.29 and 15.32 to 16.58 and from 1.69 to 3.32 and

11.14 to 15.89 respectively, The molecular sizes of W-ALB, W-GLO,W-GLU and W-

PRO analysed using SDS-PAGE under condition showed that W-ALB and W-GLO had

molecular weight between 140 and 40kDa and three major band were observed.

Thermal properties of the protein fraction provide information about their conformation

changes with temperature of denaturation. W-ALB,W-GLO,W-GLU and W-PRO had

their thermal denaturation at 82.76,92.82,91.67 and 92.43ºĊ respectively, Proteins

spectral data obtained using FT-IR and the second derivative of (1600-1700cm¯¹) this

was followed by band deconvolution that reveal the proportion of each secondary

structure making up each protein, the relative proportion are alpha helix, beta sheet, beta

turns and unordered configuration of the protein fractions varied significantly (p>0.05)

in both legume

8
3.0 MATERIALS AND METHODS

3.1 Materials

delonix regia, n-hexane, manual sieve, Bowl, Stirrer, distilled water, Funnel, NaOH,

HCl, Tray, Buffer solution, Centrifuge tube,

3.2 Equipment And Apparatus

Rotary evaporator (japan ) , laboratory blender (), centrifuge (), pH meter, weighing

balance(),

3.2 Sampling and sample preparation

Delonix regia (Flame of the forest) popularly known as sekeseke was picked from the

premises of The Federal Polytechnic Ilaro, Ogun State, the pod was remove, washed,

sun-dried and grinded into powder form using a laboratory blender () it was sieved to

obtain particles of uniform size.

3.3 Sample Preparation

The sample was extracted and defatted using the n-hexane and the oil was recovered

using the rotary evaporator. The defatted sample was sun-dried and kept in a clean

container.
9
3.4 Defatting

One Kilogram of the sample was defatted with 2.5litres of n-hexane in a batch process

and the oil was recovered using rotary evaporator.

3.5 Protein Extraction

The protein was extracted from the defatted sample using alkaline method, 1 kilogram

of defatted D. regia sample was poured into 2 L of distilled water and was stirred for

about one hour with a glass stirrer. The dispersion was adjusted with NaOH. The slurry

was then centrifuge for 30mins at 15,000rpm to remove the insoluble, After the

centrifugation the supernatant was collected and the residue was re-extracted twice

following the same procedure and the residue was discarded which is carbohydrate, the

process was repeated using the same procedure and the pH was adjusted to basic 8.0

with NaOH, insoluble non protein fraction was separated from protein extract using

centrifuge in order to increase the yields, the extraction and centrifugation process was

repeated twice on the solid residue .The protein extract (supernatant) collected was

acidified with HCl, it was stirred continuously until it reached pH 4.5 the protein extract

with HCl was washed with distilled water and was then centrifuge to eliminate all

soluble component, the pellet was washed with distilled water the protein was

suspended in distilled water maintaining total solid and then pH was adjusted to 7.0 it

was centrifuge again ,then sticky cleared protein suspension was obtained.

10
3.6 Lyophilization of the Extracted Protein sample (Freeze Drying)

The extracted Protein was first frozen for 24h at 18ºC. Frozen samples were then freeze-
dried to remove the bulk water from a freezed protein solution by sublimation under
vacuum with gentle heating (primary dry). The heating was controlled to a more
elevated temperature for removal of the remaining bound water from the protein
preparation (secondary drying). The residual moisture level are often lower than (1%)
the freeze drying operation was carried out correctly and the protein preserved all Seal
ed dialysis membrane, it was immersed in a selected buffer and the sample was loaded
using dialysis tube, it was dialyzed for about 1-2h (room temperature). The dialysis
buffer was changed and it was later dialyzed overnight (4ºc).

3.7 SDS-PAGE Gel Electrophoresis

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed to

investigate the protein profile of the samples. The gels were run under both reducing

and non-reducing conditions. A mini-slab unit was used for the electrophoresis

acrylamide concentration of the resolving gel and stacking gel was % and %

respectively. A pre-stained standard < was used as a protein molecular weight marker.

For the sample preparation, Delonix regia dispersion of % (w/v) in phosphate buffer

was stirred using magnetic stirrer for 30mins, It was centrifuged at 17500g for 30mins

and only the supernatant was loaded into the gel. The gel was run at a constant voltage

of 80 and 125Mv for stacking gel and resolving gel respectively until the dye front

reached the bottom of the frame. Gel was stained with 0.1% massie brilliant blue

followed by staining in water using methanol and acetic acid solution until the

background was clean.

3.8 FTIR (Fourier Transform Infrared Spectrometer)

11
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 CHAPTER FOUR

4.0 RESULT AND DISCUSSION

5.0 CONCLUSION AND RECCOMMENDATION

13
 REFERENCES
Ahmed, O. W., Nicholas, M., Donal, M. O. S., & Paola, T. (2022). Seed Development and
Protein Accumulation Patterns in Faba Bean (Vicia faba, L.). Journal of
Agriculture and Food Chemistry, https://doi.org/10.1021/acs.jafc.2c02061
Aluko, E. A., & Adebiyi, A. P. (2011). Functional properties of protein fraction obtained
from commercial yellow field pea (Psiumsatiouml). Seed protein isolate. Food
Chemistry, 128, 902-908.
Ayodele, O.M., & Ade-Omowaye, B. I. O. (2015). Some functional and physical
properties of selected underutilised hard-to-cook legumes in Nigeria. American
Journal of Food Science and Nutrition, 2(5),73-81.
Boye, J., Zare, F., & Pletch, A. (2010). Pulse proteins: Processing, characterization,
functional properties and applications in food and feed. Food Research
International, 43(2), 414-431.
Claudine, F., Diedericks, Carol Shek, Victoria, A., Jideani, Paul Venema, & Erik van der
Linden. (2019). Physicochemical properties and gelling behaviour of Bambara
groundnut protein isolates and protein-enriched fractions. Food Research
International, 138, 109773.
Cosson, A., Oliveira, L., Correia, Descamps, N., Saint-Eve, A., & Souchon, I. (2022).
Identification and characterization of the main peptides in pea protein isolates
using ultra high-performance liquid chromatography coupled with mass
spectrometry and bioinformatics tools. Food Chemistry. 367, 130747.
Heuzé V., Tran G., Lebas F., 2020. Flamboyant (Delonix regia). Feedipedia, a
programme by INRAE, CIRAD, AFZ and FAO.
https://www.feedipedia.org/node/308

Joshi, M., Adhikari, M., Aldred, P., Panozzo, J.F., & Kasapis, L., (2011).
Physicochemical and functional properties of lentil protein isolates prepared by
different drying methods. Food Chemistry, 129, 1513–1522.

Lawal, O. S., Adebowale, K. O., Ogunsanwo, B. M., Sosanwo, O. A., & Bankole, S. A.
(2005). The functional properties of globulin and albumin protein fractions and
flour of African locust bean (Parkia biglobossa). Food Chemistry, 92:681-691.

Luis, C. G., Adriana, A. S., Santiago, G. T., Gloria, D., Marıa, C., & Anon, (2007).
Physicochemical and structural characterization of Lima Bean (Phaseolus
lunatus) globulins. Society of food science and technology,
doi:10.1016/j.lwt.2006.11.014

Mohammad, U. M., Sabo, A. M., Roselina, K., (2017). Fractionation, physicochemical,

14
 and structural characterization of winged bean seed protein fractions with
reference to soybean Yogeshini Ramakrishnan & Kharidah Muhammad.
International Journal of Food Properties. 2, 2220-2236,
DOI:10.1080/10942912.2017.1369101

Oyedeji, O.A., Omoyeni, O. & Afuye, K., (2019). FUNCTIONAL PROPERTIES OF

PROTEIN ISOLATES FROM THE SEEDS OF Delonix Regia (FLAME OF
THE FOREST) AND Lonchocarpus Sericeus (SENEGAL LILAC). 1st National
Conference of WITED, Ilaro Chapter. The Federal Polytechnic, Ilaro. 1 (7) 471 -
477

Rojas-Sandoval, J.; Acevedo-Rodriguez, P., 2013. Delonix regia (Flamboyant). Invasive

Species Compendium, CABI Publishing, Wallingford, UK.
https://www.cabi.org/isc/datasheet/18521

Sandra, R. L., Klara, N., Jon, T., Maud. L , Jaume, G., Rafael, B., Daniel, G., & Rosana,
M., (2021). Faba bean protein films reinforced with cellulose nanocrystals as
edible food packaging material. Food Hydrocolloids.
https://doi.org/10.1016/j.foodhyd.2021.107019.

Shahidi, F., Chavan, U. D., & Mckenzies, B. B. (2001). Functional properties of protein
isolate from beach pea (Lathyrus maritimus L). Food Chemistry, 74:177-187.

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