ISOLATION OF MICROORGANISMS FROM IRU

SAMPLE IN ESA-OKE MARKET

AKINTARO TEMITOPE MARVELOUS

MATRIC NUMBER: 2211920034

BEING A PROJECT SUBMMITTED TO THE

DEPARTMENT OF MICROBIOLOGY, FACULTY OF APPLIED
SCIENCES, OSUN STATE COLLEGE OF TECHNOLOGY, ESA-OKE.

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF HIGHER NATIONAL DIPLOMA (HND) IN MICROBIOLOGY
DEPARTMENT OSUN STATE COLLEGE OF TECHNOLOGY, ESA – OKE
OSUN STATE, NIGERIA.

SEPTEMBER, 2024
DEDICATION
This project is dedicated to God Almighty and my lovely parents, Mr. and Mrs AKINTARO for
their support, kindness and words of advice toward the success of my academic pursuit.

DECLARATION
This is to declare that this project was written by me AKINTARO TEMITOPE
MARVELOUS, with Matric no: 2211920034 of Department of Microbiology, Faculty of
Applied Sciences, Osun State College of Technology, Esa-Oke.
 AKINTARO TEMITOPE MARVELOUS

Matric No: 2211920034

CERTIFICATION
This is to certify that this project was carried out by AKINTARO TEMITOPE MARVELOUS
, with Matric no: 2211920034 for the award of Higher National Diploma, Osun State College of
Technology, Esa-Oke, Osun State.

___________________ __________________
Mrs. Bamidele Date
Project Supervisor

___________________ ___________________
Mr. agbesanya Date
Head of Department

_________________ ____________________
External Examiner Date

ACKNOWLEDGEMENTS
I give the Almighty God all the glory for his provision and protection towards my life even up to

this moment, through every phase and journey of my life. He has done exceedingly abundantly

more I can ever ask for.

I am extremely grateful to my supervisor, Mrs. Bamidele for her invaluable advice, continuous

support and patience during the research period. Her immense knowledge and experience have

encouraged a lot, both research and daily life, Almighty God will continue to bless her.
My appreciation also went to the Head of the Department, Mr.Agbesanya, for his help and

support during my course of study. In the same vain, l extended my appreciations to all the

academic staff and the non-teaching staff of the department. May the great God take care of

them.

I am especially grateful to my parents Mr & Mrs Akintaro who supported me emotionally and

financially. I always knew that you believed in me and wanted the best for me. Thank you for

teaching me that my job in life was to learn, to be happy, and to know and understand myself;

only then could I know and understand others. You will reap the rewards of your labour in good

health and sound mind. I love you so much, you are simply the best. I would like to express my

gratitude to my lovely brother , sisters for his invaluable assistance.

I would like to express my gratitude to my family who have contributed to the successful

completion of this project work i pray that God reward you all.

TABLE OF CONTENTS
Title Page i
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables ix
List of Figures x
Abstract xi
CHAPTER ONE
1.0 Introduction 1
1.1 background of the study 1
1.2 problem of the statement 3
1.3 objectives of the study 3
1.4 research question 3
1.5 justificattion of the study 4
1.6 scope of research 4
CHAPTER TWO
2.0 Literature Review 5
2.1 origin of locust beans 5
2.2 African Locust Beans Seed 6
2.3 Healthy Benefits of locus beans 7
2.4 Processing of Locust Beans 8
2.4.1 Fermentation 9
2.4.2 Fermentation process 10
2.5 previous research on locust beans seed 11
CHAPTER THREE
3.0 Materials and Methods 13
3.1 Sample Collection 13
3.2 Sample pretreatment 13
3.3 Preparation of Sample 13
3.4 Fermented Seed 13
3.5 Proximate Analysis 13
3.6 Proximate Analysis of locust beans 14
3.6.1 Determination of moisture content 14
3.6.2 Determination of Ash content 14
3.6.3 Determination of Carbohydrate 15
3.6.4 Crude Protein 15
3.6.5 Determination of Crude fat 15
3.6.6 Detemination of Crude Fibre 16
3.6.7 Quality Control 17
3.7 Microbial Analysis 19
CHAPTER FOUR
4.0 Results 25
CHAPTER FIVE
5.0 Discussion and Conclusion 35
5.1 Discussion 35
5.2 Conclusion 37
References
 CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

Locust bean (Iru, Dawa dawa) is one of the most popular and widely accepted African food

condiments, and the most popular food condiment in the region of West and Central African

(Campbell-Platt, 2000), the popularity and acceptance of Locust bean was not in doubt as it is

used in soups by communities in several parts of the country. Iru-a condiment produced from the

fermentation of the dried, dehulled boiled seeds of African locust beans tree- Parkia biglobosa,

which is a perennial leguminous tree of the sub-family Mimosoideae family Leguminosae, it is

an indigenous tree with economical and social importance to the African people.

Proximate analysis refers to the determination of the major constituents of feed and it is used

to assess if a feed is within its normal compositional parameters or somehow been adulterated.

This method partitioned nutrients in feed into 6 components: water, ash, crude protein, ether

extract and crude fibre.

Locust beans serves as a rich source of plant protein to man with low cost. It also serves as good

source of protein for animal feeds, chick and fish (Livestock) (Ademola et al., 2011). Iru serves

as dietary protein in many rural areas in developing countries since some of them cannot afford

animal protein because they are either too expensive or simply unavailable. This situation has

made many people to depend mainly on carbohydrate diets; comprising of grains or starchy roots

and tuber crops with low protein level or content, thus leading to high level of malnutrition. In

the quest of rural dwellers to increase the protein level of their food, many wild fruits have been
found to be good alternative. Consumption of mainly cereal grains or starchy roots and tuber

crops leads to various health problems associated with protein and vitamin / mineral deficiencies.

In the search for plant protein and vitamin substitutes, the African locust bean (Parkia biglobosa)

has found very popular use especially in the form of fermented ‘Iru’, which is a product of its

seeds, (Gernal et al., 2007).

Apart from the nutritional values, fermented African locust bean seeds provide dietary fiber,

energy, minerals and vitamins (Vitamin B, riboflavin and Vitamin A), it also improve sensory

properties of foods which includes the organoleptic characteristics (appearance, aroma and

flavor) (Kolapo et al., 2007). One of the major benefits of fermentation is the conversion of

sugars and other carbohydrates to usable end products. Other reasons were developed as time

goes by, such as the removal of anti-nutritional components, increasing shelf life or storage,

reduction in cooking time, detoxification, decrease in the need for refrigeration or other form of

food preservation technology, enhancement of nutritional and organoleptic value of the food

(Aroma, appearance and flavor), elimination of beany flavours, improvement in digestibility and

improved safety (absence of toxins). All these benefits were brought about by microorganisms

which perform the greatest role in different parts of West Africa. There are thousands

microorganisms both inside and outside of our bodies (William and Akiko, 2004). There seems

to be a general agreement on the spore-forming Bacillus species as the main fermentation

organisms (Sanni, 2003). This was supported by Omafuvbe et al., 2002; Ouoba et al., 2002,

2003, 2004. During the fermentation of African Locust bean seeds, thorough and systematic

investigation showed that Bacillus subtillis is the most dominant bacterium responsible for the

fermentation (Antai and Ibrahim, 2006). Literature revealed that some species of bacillus such
as Bacillus lichenioformis, Bacillus megaterium, L. mesenteriodes and Staphylococcus are also

found in the fermented condiment (Iru).

In Africa many species of trees serve as sources of food and for medicinal purposes to

indigenous people. Some of these trees provide ecological services including microclimate

amelioration and soil fertility. They serve as source of income for many poor people in the rural

areas; one of these trees is Parkia biglobosa (African locust bean tree) (Igba in Yoruba

language). Farmers manage and protect this tree for their nuts and fruits. The tree has been used

both locally and internationally in drug manufacturing and cosmetics production. Despite the

important uses, the populations of this tree is reducing and it remain semi- or undomesticated

(Teklehaimanot, 2004).

However, some physical characteristics of locus beans are modified at different process stages to

increase the level of some nutrients and bioavailability (WHO, 2008) and impact some microbial

properties (Mensah et al., 2000). Processing of locus beans reduces toxicity, improve palatability

and impact desirable flavor. Processing of locus beans has also led to the development of some

essential micronutrients which are needed during the periods of rapid growth such as infancy and

adolescence (Odumodu, 2007). To improve the nutritional quality and organoleptic acceptability

of locus beans, studying the proximate composition of different processing stages of locus beans

becomes essential. This research therefore aimed at studying the effects of different processing

stages of locus beans on nutritional quality of African locust.

1.2 Statement of the Problem

During the Nigeria processing of iru, salt is usually added to prevent microbial contamination

and growth. However, inspire of these procedures iru could still harbor loafs of microorganisms
especially while handling. Hence, there is need to microbiologically evaluate the iru in other to

be ascertain its safety so as co dent used in food.

1.3 Aim and Objectives

This research work aimed at examining the proximate analysis of microbiological evaluation of

iru( locust beans condiments) sold in south west Nigeria.

1.4 Research Questions/Hypothesis (Optional)

i. What is locust bean condiments?

ii. How are they produced

iii. Why the need to microbiologically evaluate it?

1.5 Significance of the study

They also believe that the natural flavor and aroma in the traditional Iru are lost in Dawa

dawa cube. The awareness of the advantages of eating food products with little or no chemical

food additives or preservatives, with respect to human health is on the increase. It is therefore

necessary to study the different stages as they affect the quality of Iru. It is for these reasons that

this study was conducted with the aim of carrying out the common methods of processing Iru,

employing hygienic measures thereby determining the effects of the methods on the physical and

nutritive qualities of the final product.

1.6 Justification of the Study

In Nigeria, the production of locust bean has remained a traditional family art practiced in

homes especially in the rural areas with rudimentary utensils. The methods used vary from

one locality to another depending on the culture of the people, their beliefs, taste and the

practice of the fore parents who were involved in the same vocation. These variations in the
 processing techniques in turn bring about variations in the quality of Iru. Many urban

dwellers, according to field survey though are often cautious in consuming the fermented

locust bean, being skeptical about the processing hygiene, they prefer the traditionally

processed type to the industrially processed type called ‘Dawa dawa cube’ manufactured by

Cadbury Nigeria PLC because of their belief that the traditionally processed type contains

less addition of chemical preservatives than the other type.

1.7 Scope of the Study

The study intends to analyze the proximate analysis i.e moisture, ash, crude protein, ether extract

and crude fibre of different stages of locust beans production.

 CHAPTER TWO

LITERATURE REVIEW

2.1 Origin of Locust Beans

Parkiabiglobosa is a perennial deciduous tree with a height ranging from 7 to 20m,

although it can reach 30m under exceptional conditions. Crown large, spreads wide with

branches low down on a stout bole; amber gum exudes from wounds; bark dark grey brown,

thick, fissured. Leaves alternate dark green, bipinnate to 30cm long, pinnae up to 17pairs with

13-60pairs of leaflets, 8-30mm * 1.5-8mm, of distinctive shape and venation. Leaflets held on a

long rachis. Peduncles 10-35 cm long; capitula 4.5-7 cm long and 3.5-6 cm in diameter,

biglobose but distal portion much larger. Hermaphrodite flowers orange or red in colour: calyx

10-13 (16 max.) mm; corolla 10-14 (17 max.) mm long, lobes very short 1-3 mm long, connate

in the middle and free or connate at base; filaments exserted about 4 mm beyond calyx mouth.

Nectar-secreting flowers: calyx about 6-7 mm long. Staminodial flowers: calyx about 5.5-7 mm

long, filaments exserted 2-3 mm beyond calyx mouth.

The tree requires an altitude of about 300 metres with an average rainfall of 400 - 700

millimetres per year and an average mean annual temperature of 28 ⁰C. It prefers well-drained,

deep, cultivated soils, but can also be found on shallow, skeletal soils and thick laterites

(Database.prota.org, 2014). The African locust bean seeds are contained in branches of pods that
make up the most valuable part of the plant. The pods are flat and large/irregular cluster of up to

30 seeds (Omafuvbe et al., 2004). It is a tree that has spreading branches with a fat bole. It has

long tap roots and wide lateral roots up to 10 m spreading from the bole. It has a thick dark grey-

brown and a narrow opening bark from which, amber gum comes out gradually in drops when

the bark is wounded. It has dark green leaves interchange repeatedly and regularly with one

another, they are bipinnate with pinnae of up to 8-16. The tree can also grow on rocky slopes,

stony ridges or sandstone hills. Parkia biglobosa occurs in a diversity of zones, ranging from

tropical forests with high and well-distributed rainfall to arid zones where mean annual rainfall

may be less than 400 mm (Agroforestry database, 2014). Due to its deep taproot system and an

ability to restrict transpiration it has a capacity to withstand drought conditions. Parkia

biglobosa can be seen in the savannah region of Nigeria, although they are not cultivated

normally. The tree can be found mainly in Africa, especially in the following countries; Benin,

Burkina Faso, Cameroon, Chad, Cote d'Ivoire, Democratic Republic of Congo, Gambia, Ghana,

Guinea, Guinea-Bissau, Mali, Niger, Senegal, Sierra Leone, Sudan, Togo, and Uganda

(Campbell-Platt, 2000).

2.2 African Locust Bean Seed

The unfermented seed is known as karwa in Hausa; Iyere in Yoruba; Soumbala in

Burkina Faso, Mali, Cotdeivoire and Guinea. They are traditionally used as food condiment and

are known to be rich in protein and contain easily digestible calcium, they also contain 20%

edible oil. In the third world countries where the need for protein supplementation is high for

both adult and infants, ‘Iru’ is very important (Emujiugha, 2005). African locust bean (Parkia

biglobosa) seed grows on a common perennial leguminous tree known as African Locust bean

Tree which belongs to the sub-family mimosoideae and family leguminosae (now family
fabaceae), (Abdoulaye, 2012). They grow in the savannah region of West Africa up to the

southern edge of the Sahel zone 13 °C, (Campbell-Platt, 2000). The plant produces brownish

seeds, which are arranged in pods. When processed, the seeds constitute an important condiment

that adds taste and flavor to soup. The processed cake, known as ‘Iru’ is used widely in the

south-western and middle-belt zones of Nigeria. The pods, which are slightly flattened and

slender, turn from pink-brown to dark brown when mature. These pods are about 45 cm long and

2 cm wide and may contain up to 18 seeds embedded in a yellowish fleshy endocarp. The seeds

have a hard testa (averagely weighing about 0.26 g/seed) with large cotyledons forming about 70

% of their weight. Locust bean fruit is normally processed into food condiment, which is

popularly taken in the western part of Africa and it is used as a spice that gives an African meal a

pleasant flavor. Since the last 10 years they are considered as the most important food

condiments consumed everywhere by rural poor people, (Azokpota et al.,2005).

African locust bean (Parkia biglobosa) can be processed to produce afitin, iru and sonru,

three different types of condiment (Azokpota et al., 2006). ). Locust bean is generously added

to \soups as low-cost meat substitute by low-income families in parts of Nigeria, (Omafuvbe et

al., 2004). The technique used in the harvesting of locust bean is by the use of a hooked light

pole. The farmer climbs up the tree and leans on bigger branches and stretches out the hooked

pole to reach every bunch.

2.3 Healthy Benefits of Locust Bean (Iru)

Locust bean, commonly referred to as iru by Yorubas, ‘ogiri', ‘dawa dawa’ by Igbos, is a

local seasoning or condiment used in soups and stews. A very popular soup ingredient, globally,

it is referred to as African locust bean with the botanical name as Parkia biglobosa. It can be
found in a wide range of environments in Africa and is primarily grown for its pods that contain

both a sweet pulp and valuable seeds.

The yellow pulp, which contains the seeds, is naturally sweet “and is processed into a valuable

carbohydrate food known as sikomu and dawa dawa among the Yoruba and Hausa people

respectively. The most valuable parts of the locust bean are high in lipid (29%), protein (35%),

carbohydrate (16%), and is a good source of fat and calcium for rural dwellers.

The seed is first cooked to remove the seed coat and then fermented to produce the

desired result. When it is fermented, the Yoruba have a way of getting two types from it, the

mashed type and the loose or free type, and they are used for different types of soups, but for the

same purpose.

1. The fermented locust bean seed is used in controlling diabetes and cholesterol level.

2. It helps to promote good sight and aids digestion.

3. It is used for treating stroke and hypertension.

4. The water and alcoholic extracts of fermented locust bean is used to reduce blood sugar.

5. It is used in the management of bacterial infections.

6. The locust bean contains tannins, which is often recommended for the treatment of diarrhoea.

7. It is a potential benefit for enhancing weight loss.

 The crushed bark of the locust bean tree has also been revealed to help in wound healing

and serves as one of the ingredients used in treating leprosy. The decoction of the bark is also

used as bath for fever and as a hot mouth wash to steam and relieve toothache in Cote d’Ivoire.

2.4 Processing of Locust Bean

The method of preparing locust bean will be employed from the commercial producers in Ibadan

African locust beans seed

Cleaning /washing

Cooking for 6 hours

Parboiling for 1hour

Sieve

Pour into basket lined with jute Sack

Covered tight to prevent heat escape

Fermented while still hot in a dark, warm place (72hours)

Fermented Iru
Fig 1: Traditional processing of fermented African locust beans
2.4.1 Fermentation

Fermentation is a metabolic process that converts sugar to acids, gases or alcohol. It

occurs in yeast and bacteria and also in oxygen-starved muscle cells, as in the case of lactic acid

fermentation. Fermentation is also used more broadly to refer to the bulk growth of micro-

organism on a growth medium, often with the goal of producing a specific chemical product.
French microbiologist Louis Pasteur is often remembered for his insights into fermentation and

its microbial causes. The science of fermentation is known as zymology.

Fermentation takes place in the lack of oxygen (when the electron transport chain in

unusable) and becomes the cell’s primary means of ATP (energy) production. It turns NADH and

pyruvate produced in the glycolysis step into NAD + and various small molecules depending on

the type of fermentation. In the presence of O2 NADH and pyruvate are used to generate ATP in

respiration. This is called oxidative phosphorylation, and it generates much more ATP than

glycolysis alone. For that reason, cells generally benefit from avoiding fermentation when

oxygen is available, the exception being obligate anaerobes which cannot tolerate oxygen. The

first step of glycolysis is common to all fermentation pathways:

C6H12O6 + 2NAD++2ADP+2Pi 2CH3COCOO-+NADH2+2ATP+2H2O+2H+

Pyruvate is CH3COCOO-, Pi is phosphate and two ADP molecules are converted to two ATP and

two water molecules via substrate-level phosphorylation. Two molecules of NAD+ are also

reduced to NADH2.

In oxidative phosphorylation, the energy for ATP formation is derived from an

electrochemical proton gradient generated across the inner mitochondrial membrane (or in the

case of bacteria, the plasma membrane) via the electron transport chain. Glycolysis has substrate

level phosphorylation (ATP directly at the point of reaction).

Humans have used fermentation to produce foods and beverages since the Neolithic age.

For example, fermentation is used for preservation in a process that produces lactic acid as found

in such sour foods as pickled cucumbers, kimichi and yoghurt (see fermentation in food
processing), as well as for producing alcoholic beverages such as wine (fermentation in wine

making) and beer.

pH Determination:

2g of sample was weighed and suspended in 50ml of distilled water and mix thoroughly. The pH

was measured by inserting the pH probe in the solution.

2.4.2 Fermentation Process

The dried African locust bean seeds will be boiled for 12hours and allowed to soak for

12hours in boiled water. The seed will then be removed and further soaking the beans in boiling

water for the removal of the seed coat, preferably overnight. Excess water will be obtained with

cotyledons. The cotyledons were further boiled for12hours and drained off and the seeds

removed by matching for 6hours before spreading them into calabash trays and seed with the feet

in a large wooden mortar, further covering with wooden trays. Natural fermentation is removal of

seed coat is achieved by rubbing then allowed to proceed after wrapping the covered calabash

cotyledons between the palms of the hand and trays with jute bags. The fermentation will last for

three days, thereafter washing it with water. The cotyledons are then used to produce Iru.

2.5 Previous Researches on Locust Bean Seeds

All food processes are made up of a series of steps (sometimes called “unit operations”)

which have to be followed in a particular sequence in order to make the food. If the steps are

changed, or even if their sequence is changed, the process will produce a different product. The

production of locust bean condiment (Iru), is essentially a traditional family yard practice done in
rural cottage industry mainly by woman. The harvested bean seeds mainly sold in markets to

women who processed them. Sanni (2003) also describes the seven stages of processing

operation which are: Shelling, Pre-drying, Pounding, Winnowing or Sieving, Washing, Drying

and Visual sorting.

In the studies conducted on modern method of processing locust bean, it was noted that

the boring routine practice in cooking time was reduce by use of pressure cooker which reduced

the rigor 12 hours of boiling to 2 hours. Dehuller and separator – dual purpose equipment has

drastically reduced the traditional method of production of between 4 days – 6 days to 4 hours;

having production capacity of 1500 kg (Audu et al., 2004). Table 2 shows the comparative

proximal chemical composition of iru produced locally and sample from Federal Institute of

Industrial Research Oshodi, (FIIRO) Lagos, Nigeria.

Table 2.1 Comparative proximal chemical composition of iru produced locally and sample

from FIIRO

Components(%) Unfermented locust bean Fermented locust bean FIIRO

Crude protein 30.0 44.2 45.3

Fat 15.0 28.4 31.2

Carbohydrate 4.9 16.4 15.9

Crude fiber 3.1 6.9 4.6

Ash 2.9 4.1 3.0

 Omodara and Aderibigbe (2013) of the Department of Microbiology, Faculty of Science,

Ekiti State University, and Ado-Ekiti, Nigeria, reported the following from result obtained on the

proximate analysis and pH after investigating the effects of the use of starterculture on the

quality of fermented seeds. ‘Iru’ was prepared using fourteen (14) strains of Bacillus subtilis

isolated from commercial samples of ‘iru’ as starter cultures. They observed that the pH values

of all the samples increased from 7.01 to 7.58 while that of the unfermented sample was nearly

neutral or basic. The liberation of ammonia during fermentation of protein foods is a

phenomenon observed by Odunfa and Oyewole (2004), during fermentation of ‘iru’. They

observed an increase in the moisture contents of the fermented samples over the unfermented

samples which they attributed might have been due to the addition of water during soaking,

boiling and dehulling. Their result conformed to that of Omafuvbe et al., (2004) who carried out

similar research on African locust bean. They deduced that increase in protein content of starter

culture-fermented sample might be due to the structural proteins that are integral part of the

microbial cells (Teng, 2004). The apparent increase in growth and microbial proliferation of

microorganisms in form of single cell protein of the starter culture and normal flora may account

for the observed trend in the crude protein (Holzapfel, 2002).

Oladunmoye (2007) reported the proximate composition of raw, naturally fermented and

inoculated fermented seeds as shown on Table 2.1. The moisture content was found to be higher

in the fermented samples than the unfermented. Natural and inoculated fermentation gave an
increase in the crude protein value over the unfermented sample this might be due to reduction in

crude fiber and carbohydrate in the unfermented material. There was significant reduction in the

crude fibre, fat and carbohydrate as against ash contents.

CHAPTER THREE

MATERIALS AND METHODS

3.1 Sample Collection

Raw sample of locust beans seed will be bought from Bodija market, Ibadan city oyo state

Nigeria.

3.2 Sample Pretreatment

The samples collected will be pretreated by removing unwanted particles such as dirt.

3.3 preparation of sample

Sample A: 15g of the locust beans will be weight and grinded into powered form

Sample B: After the locust beans have boiled for 8hours, 15g of the sample will be weight and

grinded into powdery form.

Sample C: After the beans have been recooked for another 12hours, 15g of the sample will be

weighted and grinded into powdery form.

Sample D: After the fermentation process, 15g of the fermented beans will be weighed and

grinded into powdery form.

All samples would be air dried at room temperature.

3.4 Fermented Seeds

The locust bean will be boiled for 12 hours and the seeds dehulled. After dehulling, the locust

bean will be cooked for 6hours and fermented for several days, dried in the oven at 40 0C and

ground with food blender.

3.5 Proximate Analysis

The moisture content, ash, crude fat, crude protein (Nx6.25) and carbohydrate (by difference)

will be determined in accordance with the methods of Association of Official Analytical

Chemists (AOAC). All proximate will be carried out using chemical of Analar grade.

3.6 Proximate Analysis of Locust Bean

3.6.1 Determination of Moisture Content

1g of the sample will be weighed into a previously weighed moisture can. The moisture can and

samples taken will be transferred into a oven set at 105 0C for 4hours to a constant weight. At the

end of the 4 hours, the moisture can and sample will be removed from the oven and transferred

to a dessicator and will be cooled for 10 minutes and then after be weighed. The moisture content

will be calculated in percentage (Owoso and Ogunmoyela, 2001).

Weight of empty crucible = W2

Weight of sample = W1
Weight of crucible + sample = W3

Weight of crucible + oven-dried sample = W4

W 3−W 4
% Moisture = × 100
W1

3.6.2 Determination of Ash Content

Procedure:

I. The crucible will be weighed in order to get its weight when empty (W1)

II. The sample will then be added to the crucible and weighed (W2)

III. The sample will then be placed in muffle furnace at 500-600oC for 3 hours

IV. After removing the sample from the muffle furnace, it will then be cooled in a dessicator

V. The crucible will then be weighed again with the dry sample (W3)

W 3−W 2
Therefore, Ash % = ×100
W 2−W 1

Where W1 is weight of empty crucible

W2 is weight of crucible + wet sample

W3 is weight of crucible + dry sample

3.6.3 Determination of Carbohydrate

3g of the samples will be collected and dried in the oven at 70 0C, ground and defatted. The

soluble sugars will be extracted with 80% ethanol (v/v) (Omafuvbe et al., 2004). The total

soluble sugars will be determined by anthrone reagent method (Morris, 2003).

Total carbohydrate = 100-[moisture + crude fat + crude protein+ ash content)

3.6.4 Crude Protein

20ml of concentrated sulphuric acid will be introduced into the micro-kjeldahl flask containing

3grams of ground sample. Using the Kjeldahl methods, three steps are involved, which include;

digestion, distillation and titration.

Digestion

3g of the sample will be weighed into the Kjeldahl flask and 20ml of conc. H 2SO4, 0.8g of

CuSO4, 6g Na2SO4 and speck of selenium tablet will be added. Heat will then be applied in a

fume cupboard (because the gases that will evolve might be lacrimatory to the skin) slowly at

first to prevent undue frothing, the digestion will continue for several minutes. It will be left until

it cools completely and 100ml of distilled water will be added. The digestion flask will be rinsed

thoroughly and it will be poured into the bulk.

Distillation

Markham distillation apparatus will be used for the distillation process. The apparatus will be

steamed up and 15ml of the digest will be put into the apparatus through a funnel and will be

allowed to boil. 10ml of NaOH will be added so that any gas produced will not be lost. The

whole mixture will be distilled into 60ml of 3% boric acid using a suitable indicator.

Titration

The alkaline borate formed will be titrated directly with 0.1M HCL. The volume of the acid used

will be recorded. It is this volume that will be used to calculate the total proteins in the locust

bean.

%N = ¿ ¿
VA = volume of acid used W= Weight of sample

% Crude Protein = %N x 6.25

3.6.5 Determination of Crude Fat

1g of each sample will be weighed into a fat free flask. Another flask will be dried in the oven,

25ml of n-Hexane will be added to each samples in the flask and will be stirred, then it will be

heated in the water bath at 6070C for exactly 5 minutes and will be removed and stirred. The

dried flasks will be weighed as W2 and the content of the first flask will be filtered into it. The

filtrate will then be dried in the oven until all solvents evaporated and the flask will be cooled

and then reweighed as W3.

Weight of sample = W1

Weight of dry flask = W2

Weight of sample after evaporation of solvents = W3

W 3−W 2
% Fat = ×100
W1

3.6.6 Determination of Crude Fibre

1g each samples will be measured into the fibre flask and 100ml H 2SO4 will be added. The

mixtures will be heated under reflux for 1 hour with the heating mantle. The mixtures will be

filtered through a fibre sieve cloth. The filtrates obtained will be thrown off and the residues will

be returned to the fibre flask to which 100ml of 0.313N NaOH will be added under reflux for

another 1 hour. The mixtures will be filtered through a fibre cloth and 10ml of acetone will be

added to dissolve any organic constituent. The residues will be washed with about 50ml hot
water on the sieve cloth before they will be finally transferred into the crucible. The crucible and

residue will be oven-dried at 105 0C overnight to drive off moisture. The oven-dried crucible

containing the residue will be cooled in a dessicator and later weight W 2. The crucible with

weight W1 will be transferred to the muffle furnance for ashing at 5200C for 4 hours.

The crucible containing white or grey ash (free of carbonaceous material) will be cooled in the

dessicator and weighed to obtain W 3. The difference W2-W3 will give the weight of fibre. The

percentage will be calculated below

Weight of crucible = W1

Weight of oven-dried crucible + residue = W2

Weight of crucible + ash after cooling = W3

W 2 −W 3
% fibre = × 100
W t of Sample

3.6.7 Quality Control

i. The sample was weighed for each stages of the production

ii. The Sample was parboiled very well

3.7 MICROBIAL ANALYSIS:

3.7.1. Media Preparation:

The media used were Nutrient Agar, MacConkey Agar, Potato Dextrose Agar and Peptone water

Broth. These media were prepared in accordance with the method of AOAC (1990).

3.7.2. Identification of Isolated Cultures:

Bacteria identification was based on colony characteristics, cellular morphology and biochemical

reactions as described by Uzuegbu et. al., (2001). While fungi identification was based on their

cultural and morphological characteristic.

3.7.3. Bacteria Identification Tests:

After morphological examination of the colonies, all bacteria isolated were subjected to the

following biochemical tests: gram staining, catalase, oxidase, coagulase, indole, citrate, motility

and sugar fermentation tests.

3.7.4. Gram Staining Test:

The smear of the isolates BA, BB1, BB2, BC, BD1 and BD2were each prepared on clean glass

slides. They were heat-fixed by passing them over Bunsen flame three times. The smear on the

slides were then flooded with crystal violet stain for 60secs. This was washed off under running

tap water. The smears were then flooded with iodine solution for 30sec and rinsed with running

water, and then decolourized with 95% ethyl alcohol. After washing off the alcohol, the smears

were counter stained with safranin for 30sec and washed off under running water. They were

blotted dry with clean blotting paper and examined under oil immersion with objective lens of

the microscope. Purple colouration of bacteria indicates that is gram positive while the red

colouration indicates gram negative.

3.7.5. Catalase Test:

This test is to determine the microorganisms that contain catalase enzyme capable of releasing

oxygen gas when mixed with hydrogen peroxide (H2O2). A drop of 3% hydrogen peroxide was

placed on a clean grease-free slide. A loop full of 24h old culture was placed on it and examined

for the appearance of bubbles. The evolution of gas bubbles indicates positive catalase reaction
3.7.6. Oxidase Test:

Some organisms have the tendency of forming a complex mixture with the oxidase reagent

(Para-amino dimethyl phenylenediaminemonochloride and water) and appearing purple on filter

paper soaked with the reagent. Few drops of freshly prepared oxidase reagent were placed on

sterilize filter paper. Test cultures (24h old) were streaked on the filter paper soaked with the

reagent. The appearance of a purple colour after 10secs indicates positive reaction.

3.7.7. Coagulase Test:

the slides were then flooded with crystal violet stain for 60secs. This was washed off under

running tap water. The smears were then flooded with iodine solution for 30sec and rinsed with

running water, and then decolourized with 95% ethyl alcohol. After washing off the alcohol, the

smears were counter stained with safranin for 30sec and washed off under running water. They

were blotted dry with clean blotting paper and examined under oil immersion with objective lens

of the microscope. Purple colouration of bacteria indicates that is gram positive while the red

colouration indicates gram negative. This test differentiates Staphylococcus aureus which

produce the enzyme coagulase from the non-coagulase producing Staphylococcus and

Micrococcus species. The enzyme coagulase causes plasma to clot by converting fibrinogen to

fibrin. A drop of physiological saline was placed on each end of a slide. The colony of the test

organism was emulsified on each of the drop to make two thick suspensions. A drop of plasma

was added to one of the suspension and mixed gently. It was checked for dumping of the

organisms with 10sec

3.7.8. Indole Test: This helps in the determination of the bacteria capable of breaking down the

amino acidtryptophan to indole. A 3% peptone water was prepared by dissolving 3g of the

medium in 100ml of distilled water. Then the medium was dispensed into test tubes and

sterilized at 1210C for 15mins. After sterilization the medium was allowed to cool and was

inoculated and incubated at 370C for 2-3 days to allow for the growth of the test organisms. The

presence of indole after incubation was detected by adding a few drops of kovac‟s reagent (5g-p-

dimethyl amino benzaldehyde, 57g amyl alcohol, 25ml conc. HCl) to the culture. Ring-like red

colouration on top of the tube indicates positive result.

3.7.9. Citrate Test:

The citrate test was performed by inoculating into organic synthetic medium in which sodium

citrate is the only sources of carbon and energy. In sodium citrate broth (Koser‟s citrate

medium), the presence of growth (turbidity) is a positive test result.

3.7.10. Motility Test:

A ring of Vaseline was made on a clean grease free microscopic slide. A drop of 24h old broth

culture of the isolates was placed on a clean slide cover slip. The slide was inverted quickly and

gently over the cover ship which adhered to the slide. It was immediately examined under the

microscope, for motility of organisms. The appearance of darting or rigging in the broth signifies

motility.

Sugar Fermentation Test:

The ability of organisms of ferment different sugars could be used as a distinguishing factor.

Aliquot portions of the basal medium (Peptone-sugar water) was dispensed into a test-tube and

filled by gently inversion of durham tubes into the medium. The test cultures (24h old) were then

incubated into the sterilized medium and incubated for 48h at 370C. A colour change from red to
yellow (with phenol red indicator) and air space in the durham tubes was considered positive,

indicating acid and gas production respectively.

Sensory Evaluation of The ‘Iru’ And Ogiri In Soup Sampels:

Sensory evaluation was carried out using eleven taste panelists to assess the sensory attribute

(colour, aroma, taste, texture and overall acceptability) of the produced food condiments (iru and

ogiri-iru blend).The soups prepared with the „iru‟ and „ogiri-iru blend‟ seasonings were

presented to the panellists, using the soup prepared with commercial „ogiri‟ as a reference. The

panellists were selected randomly cutting across students and workers of the university

community which include people who are used to eating „iru‟ and „ogiri‟ and those who are not

used to them. The samples were presented in coded identical plates. The panellists are instructed

to rate the sample for the parameters based on a 7- point hedonic scale ranging from 7-liked

extremely to 1-disliked extremely. The raw scores were assembled and statistically analyze using

the method described by Ihekoronye and Ngoddy (1985).

Statistical Analysis:

Data obtained from the study of the sensory evaluation was subjected to analysis of variance

(ANOVA) and the means were separated using Fisher LSD and judged significantly different at

95% confidence level (i.e P<0.05).

 CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 Introduction

A wrap of the fermenting seeds was taken at the start of fermentation and at 24h interval for 4

days. Five grams (5g) of each samples was weighed into a sterile mortar and mashed with clean

beaker and 50ml of distilled water was added. It was mixed thoroughly to form slurry. A

standard buffer solution (pH 6.0) was prepared and this was used to standardize the pH

meter (Dye Unicam,Model PW 9409). The electrode of the digital pH meter was dipped in the

slurry. The pH readings were recorded.

4.2 Presentation of Results

SAMPLE CODE NA EMB

(total bacteria count) TOTAL Col form count

NA10-3

ST 10-2 TnTc

ST 10-2 TnTc

DSI 10-2 200

DS3 10-2 172

DSI 10-3 164

MW1 10-2 TnTc

MW2 10-3 188

MW2 10-2 TnTc

WW 10-3 TnTc

WW 10-2 291

KW 10-3 TnTc

KW 10-2 TnTc

BW 10-3 TnTc

BW 10-2 TNTC

LSF1 10-3 12

LSF1 10-2 26

LSF2 10-2 31
 LSF2 10-3 4

4.3 Discussion of Findings

The results of the composition of raw locust beans seeds, first boiling (8 hours boiling), second

boiling (20 hours boiling) and the fermented locust beans are shown in the table 4.1 above

Fermented locust beans gave relatively high protein content over the raw locust beans,

the first 8 hours boiled locust beans and the second 20 hours boiled locust beans seeds. The

results obtained were 21.88%, 19.67%, 17.89% and 16.89% from the analysis of the fermented,

raw, first 8 hours boiled, and the second 20 hours boiled locust beans respectively. These results

were in correlation with that of Kolapo et al., (2007), which observed a low protein content in

three samples. Protein is an essential component of diet which supplies adequate amount of

amino acids (Pugalenthi et al., 2004). The cause for this reduction in the protein content in boiled

samples might be as a result of the heat applied during the preparation, because heat denatures

protein. The high content of protein in fermented samples might be as the result of the amount of

water used for fermentation which might have caused some enzymes or micro- organisms to

inhibit the protein greatly. The cause for low protein content in the raw seeds was probably due

to the low level of water and this might inactivate some enzymes.

The crude fibre analyzed from the table above showed that raw locust beans sample has a

relatively high crude fiber content, followed by fermented locust beans, 20 hours boiled locust

beans and 8 hours boiled locust beans seed. The result obtained were 6.29%, 2.16%, 1.89% and

1.67% for raw, fermented, 20 hours boiled locust and 8 hour boiled locust beans respectively.

These results were in tandem with the work done by Hassan et al.,(2007) whose result showed
that Parkia biglobosa fruit is within the range of 3.17% to 0.29% except the raw locust bean that

has a crude fiber of 6.29%. Fibre plays an important role in the prevention of number of diseases

like reducing the level of cholesterol and also helps in the prevention of colon cancer as well as

quick digestion of foods.

The ash content ranged from 4.69% to 6.51%, with raw locust beans seeds having the

highest value and the 8 hours boiled locust beans having the lowest value. The sequence are from

4.69%, 4.87%, 5.13% and 6.51% for 20 hours boiled, 8 hours boiled sample, fermented and raw

locust beans seeds respectively. Ash content gives an idea of the inorganic content of the samples

analysed from where the mineral content could be obtained as stated by sotelo et al., (2007). The

20 hours boiled and 8 hours boiled sample did not show any significant difference, hence boiling

has little or no effect on the ash contents of locust beans seeds.

The moisture content was found to be within the ranges of 5.04% - 13.38% with raw

sample having the lowest and the 8 hours boiled having the highest value. The results obtained

are in the order of 5.04%, 6.18%, 12.38% and 13.38% for raw, fermented, 20 hours boiled and 8

hours boiled samples respectively. This showed that during the processing stages, the moisture

content increased gradually. These results were in accordance with the report of Omafuvbe et al.,

(2004) who revealed that moisture content increases during processing.

The ether extract was found to be within the range of 5.68-8.46% with 20 hours boiled sample

having the lowest and raw sample having the highest. The results obtained are in the order

5.68%, 6.52%, 7.89% and 8.46% for 20 hours boiled sample, 8 hours boiled sample, fermented

and raw sample respectively. These results showed that ether extract decreases during processing

stage. This decrement in the value of ether extract is in tandem with Owoso and Ogunmoyela,

(2004) whose work showed decreased in ether extract during processing

 CHAPTER FIVE

SUMMARY, CONCLUSIONS AND RECOMMENDATION

V.1 Conclusion

The identification of the predominant microorganisms in the fermented, iru was performed in

the present work for the first time. Diversity was observed at species level of Bacillus

(primarily in B. subtilis but also in B. licheniformis and B. cereus) as well as at strain level.

Staphylococcus spp were initially identified in afitin, but recent investigations have revealed

their presence also in iru. B. subtilis and B. licheniformis were differentiated by 16S rDNA
 sequencing and by additional biochemical testing and API 50 CHB system, illustrating the

possibility of combining genotyping and phenotypic analyses to distinguish closely related B.

subtilis and B. licheniformis species.

For further investigation, the identified Bacillus spp are potential target to be used as starters

culture to produce iru in controlled fermentation. First of all, there is a need to deeply

characterize these strains, since the final selection of strains to be used as starter culture will

depend on individual performance under practical conditions, proteolytic or lipolytic activity

and production of a desirable aroma.

5.2 Recommendation

It can be recommended that suspension of Bacillus subtilis administered orally to broiler in the

range (5.0x105 - 2.0x106 CFU/mL) used in this study significantly increased feed intake, water

intake, serum albumin, albumin-globulin ratio and cholesterol of the birds. No significant effect

of the probiotics suspension was observed on the haematology and carcass yield of the broilers

except the back, neck and abdominal fat weights. Furthermore, the relative weights of organs of

broilers monitored in this study were not significantly affected. It would appear that suspensions

of B. subtilis at the inclusion levels used in this study did not have significant effect on most
performance and carcass qualities parameters monitored, further investigation should be carried

out using B. sub

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