1

EFFECT OF DIFFERENT PROCESSING METHODS ON

THE CHEMICAL COMPOSITION OF AFRICAN YAM BEAN
(SPHENOSTYLIS STENOCARPA) FLOURS AND ORGANOLEPTIC
CHARACTERISTICS OF THEIR GRUELS.

BY

ABURIME, LILIAN CHINELO

PG/MSc/08/49360

DEPARTMENT OF HOME SCIENCE, NUTRITION AND DIETETICS

UNIVERSITY OF NIGERIA, NSUKKA

MARCH, 2012.
 i

EFFECT OF DIFFERENT PROCESSING METHODS ON

THE CHEMICAL COMPOSITION OF AFRICAN YAM BEAN
(SPHENOSTYLIS STENOCARPA) FLOURS AND ORGANOLEPTIC
CHARACTERISTICS OF THEIR GRUELS.

A PROJECT REPORT SUBMITTED IN PARTIAL FULFILMENT

OF THE REQUIRMENTS FOR THE AWARD OF MASTER’S
DEGREE IN HUMAN NUTRITION

BY

ABURIME, LILIAN CHINELO

PG/MSc/08/49360

DEPARTMENT OF HOME SCIENCE, NUTRITION AND DIETETICS

UNIVERSITY OF NIGERIA, NSUKKA

MARCH, 2012.
 ii

APPROVAL PAGE

This project report has been approved for the Department of Home Science,
Nutrition and Dietetics

By

----------------------------- ------------------------------
Prof. Mrs. E.K Ngwu Prof. Mrs. N.M Nnam
Supervisor Head of Department

Date-------------------------- Date---------------------------
 iii

CERTIFICATION

Aburime, Lilian Chinelo, a postgraduate student in the Department of Home Science,

Nutrition and Dietetics University of Nigeria Nsukka, with registration number

PG/M.Sc/08/49360 has satisfactorily completed the requirements for the award of the degree

of Master of Science in Human Nutrition. The work embodied in this project report is

original and has not been submitted in part or full for any other diploma or degree of this or

any other university.

------------------------------- ------------------------------------
Prof. Mrs. E.K Ngwu Prof. N.M Nnam
Supervisor Head of Department

Date:------------------------- Date:----------------------------

-----------------------------------
Prof. L. I. Salami
External Examiner

Date:-------------------------
 iv

DEDICATION

This work is dedicated to God Almighty.

 v

ACKNOWLEDGEMENTS

The researcher’s special thanks go first to her project supervisor Prof. Mrs. E.K Ngwu
for the corrections and guidance she gave to her at every stage of this work, most especially
her patience in reading through her work. She also thanks her for the motherly advice and
encouragements she gave her throughout her study.
The researcher is indebted to the Head of her Department Prof. Mrs. N.M Nnam
whose objective criticisms and useful suggestions and corrections were driving forces to the
successful completion of this work. She appreciates her patience in reading through this
work.
The researcher’s sincere appreciation goes to Prof. E.C Okeke and Prof. I. C Obizoba
for their countless support in the sphere of academics, which have equally contributed to the
successful completion of this program. She is also grateful to Dr. Mrs Onyechi, U.A and
other members of the Postgraduate board of this Department for the great knowledge
impacted to her and also their academic contributions towards the betterment of this work.
Her sincere gratitude goes to other academic and non academic staff of the department for
their guidance and care through out her stay in the department.
The researcher is highly grateful and indebted to her dear husband Giovanni Aburime
whose encouragement, love, care and great support has immensely led to the successful
completion of this work. She is also thankful to her little angels (Rapheal and Anabell) for
enduring the discomforts associated with her constant absence from home because of this
work and also appreciate their prayers.
The researcher will forever remain grateful to her dear mother Mrs. Victoria Nwodili
and her siblings whose care and prayers led to the successful completion of this work. She is
grateful to her parent in laws Mr. and Mrs. Edward Aburime. It’s a pity that her mother in-
law did not see the end of this work, which she supported so much, may her gentle soul rest
in peace. Amen.
She thanks all the Rev. Fathers who supported her in prayers. She thanks all her
friends, prayer partners, men and women who avail themselves to be used by God to bring
this work to a good end. May God bless you all.
Above all, she thanks God Almighty who has brought this work to a successful
completion.
 vi

TABLE OF CONTENTS

Title page - - - - - - - - - i
Approval page - - - - - - - - - ii
Certification - - - - - - - - - iii
Dedication - - - - - - - - - iv
Acknowledgements - - - - - - - - - v
Table of contents - - - - - - - - -
List of tables - - - - - - - - - vi
List of figures - - - - - - - - - vii
List of appendices - - - - - - - - - viii
ABSTRACT - - - - - - - - - xii

CHAPTER ONE: INTRODUCTION - - - - - - 1

1.1 Statement of the problem - - - - - - - - 3
1.2 Objectives of the study - - - - - - - - 4
1.3 Significance of the study - - - - - - - - 4

CHAPTER TWO: LITERATURE REVIEW - - - - -- 6

2.1 Legumes in human nutrition - - - - - - - 6
2.2 Functional properties of legumes - - - - - - - 7
2.3 Legumes in diet-related non communicable disease - - - - 8
2.4 Bean as a legume - - - - - - - - 9
2.5 African yam beans (AYB) (Sphenostylis stenocarpa ) - - - - 9
2.5.1 Nutritional/ chemical composition and organoleptic attributes of AYB - 10
2.5.2 Antinutrient composition of 3 varieties of African yam bean - - - 13
2.5.3 Economic importance/uses of AYB - - - - - - 13
2.5.4 Constraints in the use of AYB - - - - - - - 14
Hydrogen cyanide (HCN) - - - - - - - 15
Hemagglutinin (Lectins) - - - - - - - 15
2.5.5 The antinutrients in AYB - - - - - - - 15
Phytic acid - - - - - - - - - 16
Tannins - - - - - - - - - 16
Saponins - - - - - - - - - 16
Trysin inhibitors - - - - - - - - 17
Oxalates - - - - - - - - - 17
2.5.6 AYB processing methods used - - - - - - - 18
Roasting - - - - - - - - - 18
Fermentation - - - - - - - - - 18
2.6 Lime - - - - - - - - - - 20

CHAPTER THREE: MATERIALS AND METHODS - - - - 21

3.1 Materials - - - - - - - - - 21
3.2.1 Preparation of African yam bean (AYB) flour for gruel and chemical analysis 21
24h fermentation without lime and roasting - - - - - 21
24h fermentation with lime and roasting - - - - - 21
48h fermentation with lime and roasting - - - - - 21
Roasted only - - - - - - - - - 22
3.2.2 Preparation of gruel from AYB flours - - - - - 22
3.3 Laboratory analysis - - - - - - - - 22
3.3.1 Protein determination - - - - - - - - 23
 vii

3.3.2 Fat determination - - - - - - - - 24

3.3.3 Ash determination - - - - - - - - 24
3.3.4 Crude fibre determination - - - - - - - 25
3.3.5 Carbohydrate - - - - - - - - - 25
3.3.6 Phytate determination - - - - - - - 25
3.3.7 Tannins - - - - - - - - - 26
3.3.8 Determination of trypsin inhibitors - - - - - - 27
3.3.9 Oxalic acid determination - - - - - - - 28
3.3.10 Determination of saponins - - - - - - - 29
3.3.11 Hydrocyanic acid determination - - - - - - 30
3.3.12 Determination of Haemagglutinin by spectrometric method - - - 31
3.3.13 Raffinose and stachyose determination - - - - - 31
3.4 Organoleptic evaluation - - - - - - - 32
3.5 Statistical analysis - - - - - - - - 33

CHAPTER FOUR: RESULTS - - - - - - - 34

4.1 Proximate composition of different AYB flours on dry matter basis - -
4.1.1 Protein - - - - - - - - - 34
4.1.2 Fat - - - - - - - - - - 34
4.1.3 Ash - - - - - - - - - - 34
4.1.4 Crude fibre - - - - - - - - - 34
4.1.5 Carbohydrate (CHO) - - - - - - - - 35
4.2 Effect of treatments on the anti nutrient contents of AYB flour samples -
4.2.1 Phytate - - - - - - - - - 35
4.2.2 Tannins - - - - - - - - - 35
4.2.3 Oxalates - - - - - - - - - 35
4.2.4 Saponins - - - - - - - - - 35
4.2.5 Trypsin inhibitors - - - - - - - - 35
4.3 Effect of treatment on the raffinose, stachyose, heamagglutinins and hydrogen
cyanide composition of AYB flours - - - - - -
4.3.1 Raffinose - - - - - - - - - 37
4.3.2 Stachyose - - - - - - - - - 37
4.3.3 Heamagglutinins - - - - - - - - 37
4.3.4 Hydrogen cyanide (HCN) - - - - - - - 38
4.4 Organoleptic characteristics of AYB gruel - - - - -
4.4.1 Colour - - - - - - - - - 38
4.4.2 Flavour - - - - - - - - - 39
4.4.3 Consistency - - - - - - - - - 39
4.4.4 Degree of acceptability - - - - - - - 39

CHAPTER FIVE:DISCUSSION, CONCLUSION AND RECOMMENDATIONS 41

5.1 Discussion - - - - - - - - -
5.1.1 Effect of processing on the proximate composition of AYB flours - -
Protein - - - - - - - - - 41
Fat - - - - - - - - - 41
Crude fibre - - - - - - - - - 41
Carbohydrate - - - - - - - - - 42
5.1.2 Effect of treatments on the anti nutrient composition - - -
Phytate - - - - - - - - - 42
Oxalates - - - - - - - - - 42
Tannin - - - - - - - - - 42
 viii

Trypsin inhibitors - - - - - - - - 43
Effect of processing on the raffinose and stachyose contents of AYB flours 43
5.1.3 Effect of treatment on the toxic substance composition of AYB flour samples
Heamagglutinin - - - - - - - - 43
Hydrogen cyanide (HCN) - - - - - - - 44
5.1.4 Effect of treatment on the beany flavour and other organoleptic
characteristics of AYB gruel - - - - - - -
Colour - - - - - - - - - 44
Flavour - - - - - - - - - 44
5.2 Conclusion - - - - - - - - - 45
5.3 Recommendations - - - - - - - - 45

REFERENCES - - - - - - - - - 46
APPENDICES - - - - - - - - - 53
 ix

LIST OF TABLES

Table 1: Proximate (% dry matter) and mineral (mg/100g dry matter) composition
of cream, brown and brown spotted (speckled) raw AYB seeds - - 12

Table 2: Amino acid distribution of AYB - - - - - - 13

Table 3: Antinutrient composition of 3 varieties of African yam bean (raw seed

characterization) - - - - - - - - 13

Table 4: Effect of treatments on proximate composition of different AYB flour samples

on dry matter basis (%) - - - - - - - 34

Table 5: Anti nutrient composition of AYB flours (mg/g) - - - - 36

Table 6: Raffinose, stachyose, heamagglutinins and hydrogen cyanide composition of

AYB flours - - - - - - - - - 38

Table 7: Organoleptic characteristics of AYB gruels - - - - - 39

 x

LIST OF FIGURES

Figure1. Flow chart for the processing of AYB flour - - - - 23

 xi

LIST OF APPENDICES

Appendix I: Hedonic scoring form for the evaluation of AYB gruel - - 53

Appendix II: General acceptability rating of the sample - - - - 54
Appendix III: Raw cream coloured AYB seed - - - - - 55
Appendix IV: Gruels from AYB flour samples - - - - - 56
 xii

ABSTRACT

The aim of this study was to determine the effect of different processing methods on the
chemical composition of African yam bean (Sphenostylis stenocarpa) flours and the
organoleptic properties of it’s gruels. The seeds of cream coloured African yam bean (AYB)
and lime were purchased from Oye Igbo-Eze and Ogige Nsukka markets, respectively in
Enugu State, Nigeria. The seeds were sorted and divided into 4 equal portions of 1.5kg each.
One portion was washed and fermented in tap water at a seed- water ratio of 1:3 (w/v), for
24h. The second portion was fermented in tap water (1:3 w/v) containing 30 tablespoonful of
freshly squeezed lime for 24h. The third portion was fermented in tap water containing lime
(30 tablespoonful of lime) (1:3 w/v) for 48h. They were separately sundried for 72h and
roasted in a hot sauce pan until cracking. The fourth portion (control) was washed, drained
and roasted in a hot sauce until cracking. The AYB samples were separately milled into fine
flours and stored in separate airtight containers for chemical analysis and gruel preparations.
The flour samples were chemically analyzed for proximate, phytate, tannins, oxalates,
saponins, trypsin inhibitors, raffinose, stachyose, hemagglutinins and hydrogen cyanide
composition using standard laboratory methods. A nine point hedonic scale was used to
collect data on sensory and acceptability tests of the gruels. Means and standard deviations
were calculated and least significance difference test was used to separate means. The sample
that was fermented in tap water containing lime for 48h ranked best generally. The moisture
levels for the flours ranged from 3.60-5.00%, protein 19.96-31.87%, fat 3.54-5.23%, ash
2.99-3.89%, crude fibre 4.00-6.01% and carbohydrate 52.72-62.32%. The anti-nutrient values
for the flours were: phytate 2.63-2.97mg/g, tannins 0.02-0.04mg/g, trypsin inhibitors 0.45-
0.53mg/g, oxalate 0.01-0.03mg/g, the samples had the same saponin level of 0.01mg/100g.
Raffinose contents of the samples ranged from 8.25mg-9.22/100g and stachyose (8.48-
6.76mg /100g). heamagglutinins ranged from 4.87 – 6.70 mg/100g and hydrogen cyanide
ranged from 0.22-0.28mg/g. In the organoleptic studies, the sample that was fermented for
48h was most preferred over others in terms of colour (6.53), the sample that was fermented
in lime water for 24h ranked best for flavor (6.57) and the sample that was only roasted
ranked best for general acceptability (6.03).
 1

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the study

It is of great importance to know the nutrient, toxic substance as well as the anti

physiological substance composition and organoleptic properties of locally available foods in

any community or country. Knowledge and use of local foods can help eliminate malnutrition.

One of the problems of planning therapeutic diets with local foods is limited information on

their nutrient composition (Standing Committee on Nutrition (SCN), 2006). It has been

proposed that the fight against malnutrition in developing countries should be on the use of

mixtures of tubers, cereals and legumes indigenous to them (Nnam, 2003).Urbanization has

made people forget their traditional foods and favour convenient foods which are mostly

nutritionally inadequate and expensive. The most dietary deficit is protein of high biological

value and this was attributed to the high cost of animal protein (SCN, 2006). Vegetable

proteins however complement each other if well chosen and will have a nutritive value as good

as animal protein (Achinewhu & Akah, 2003; Nnam, 2003; Obiakor, 2008).

Nutrition is coming to the fore front as a major modifiable determinant of chronic

diseases, with scientific evidence increasingly supporting the view that alterations in diet have

strong effects (both positive and negative) on health throughout life. Dietary adjustments may

not only influence present health, but may determine whether or not an individual will develop

such diseases as diabetes, obesity, hypertension, certain cancer and cardiovascular disease

much later in life (WHO/FAO, 2003). Rapid change in disease pattern had occurred as a result

of shifts in diet and lifestyle. The urban based Nigerian is shifting from exercise, intense

agrarian life to a more sedentary urban life, with resultant obesity, diabetes and hypertension.

Cheap imported foods, global markets and socio-cultural changes are placing African

traditional diets at distinct disadvantages. Indigenous diets are being replaced with more

refined carbohydrate fast foods (Ifeyironwa, Eyzaquirre, Matig, & Johns, 2006). In tackling the
 2

multiple problems of food insecurity, nutrition transition and the double burden of diseases, it

is essential to mobilize and employ indigenous foods like legumes as part of the solution

(SCN, 2006). This is because several studies have reported immense nutritional and health

protecting properties of African indigenous foods such as legumes (Obizoba & Souzey, 1989;

Enwere, 1998; Ene-Obong & Carnovale, 1992; SCN, 2006; Okeke, Ene-Obong, Uzuegbunam,

Simon, & Chukwuone, 2009).

For quite some time, legumes were considered not too important; but now, their food

use is increasing with recent discoveries concerning their many nutritional and health

properties (Pamplona-Roger, 2006).It has been documented that legumes contain 2-3 times the

protein of cereal grains and no other plant food is as rich in protein as legumes in their natural

state (National Academy of Science (NAS), 1997; Pamplona-Roger, 2006). Water soluble non-

starch polysaccharides (NSP) that have viscous properties occur mostly in legumes and its

benefit in the prevention/management of diabetes and cardiovascular diseases have been

reported (Onyechi, Jude, & Ellis., 1998; Enwere, 1998). One such legume of interests is

African yam bean (Sphenostylis stenocarpa) (AYB).

African yam bean (AYB) is an herbaceous leguminous plant occurring throughout

tropical Africa (United States Department of Agriculture (USDA), 2007). It is grown as a

minor crop in association with yam and cassava. AYB serves as security crop; it has the

potential to meet year round protein requirements if grown on a large scale (World Health

Organization (WHO), 2002). African yam bean (AYB) is highly nutritious with high protein,

mineral and fibre content. Its protein content is reported to be similar to that of some major and

commonly consumed legumes. Its amino acid profile is comparable if not better than those of

cowpea, soy bean and pigeon pea (Obizoba & Souzey, 1989; Ene-Obong & Carnovale, 1992;

Uguru & Madukaife, 2001). It has high metabolic energy, low true protein digestibility

(62.9%), moderate mineral content, the amino and fatty acids contents are comparable to those
 3

of most edible pulses (Nwokolo, 1987; Uguru & Madukaife, 2000). It has a higher water

absorption capacity when compared to cowpea (Achinewhu & Akah, 2003).

The potential role of AYB in the management of many aging and chronic non-

communicable diseases has been reported (Enwere, 1998; Nwachi, 2007; Alozie, Udofia,

Lawal & Ani, 2009). In Ghana, the water drained after boiling may be drunk by lactating

mothers to increase their milk production (Klu, Amoatey, Bansa & Kumaga, 2001). The

economic potential of AYB has been recognized, especially in reducing malnutrition among

Africans (Adewale, 2010).

These health benefits can be marred by the presence of anti- nutrients. Some processing

methods however, such as soaking, boiling, fermentation, roasting, among others are known to

achieve reduction or elimination of the anti nutritional factors which affect the nutrients

(Nnam, 1994., Nnam, 1995; Ene-Obong & Obizoba, 1995; Obizoba & Atti, 1994; Messina,

1999; Nnam, 1999).

1.2 Statement of the Problem

Studies have shown that inspite of the good attributes of AYB, it is underutilized and

rarely consumed in urban and rural areas in Nigeria. Its current status as a minor crop means

that its potential is largely unexploited. It faces the danger of extinction (Klu et al., 2001). The

use of AYB may be limited by the beany flavour and long cooking time. These limitations can

be overcome by processing like fermentation, soaking, roasting among others (Nnam, 1994;

Ene-Obong & Obiziba, 1995; Nnam, 1999; Fasoyiro, Ajibade, Omole, Adeniyan & Farinde,

2006; Adewumi and Odunfa, 2009). Moreover, soaking or fermenting in lime medium can

equally improve flavour, reduce the incidence of flatulence and acts as anti oxidant

(OnlineFamilydoctors, 2000, Waladkhani, & Clemens, 2003; NewWorldencyclopedia, 2010).

Citric acid treated AYB has been shown to have greater reduction in toxic substances like the

cyanides (Azeke, et al., 2007).

 4

Some studies have been carried out on the nutrient, anti nutrient and toxic substance

composition of AYB and some of its products and processing methods to improve the food use

(Ene-Obong & Obizoba, 1995; Nnam, 1997; Nnam, 2003; Omeire & Ogbonna, 2006; Onyechi

and Nwachi, 2008). Little has been done on the effect of 24h fermentation with and without

lim e, 48h fermentation in lime water and roasting to address the beany flavour. Little has

equally been done on the effects of these processing methods on nutrient, anti nutrients, toxic

substances, anti physiological factors and other organoleptic characteristic of AYB flour and

gruel.

There is the need therefore to determine the effect of 24h fermentation with and without

lime and subsequent roasting, 48h fermentation in lime water and subsequent roasting on the

chemical composition of AYB flours and organoleptic characteristics of their gruels.

1.3 Objective of the study

General objective

The general objective of the study was to determine the effect of different processing

methods on the chemical composition of AYB flour and organoleptic characteristics of their

gruels.

The specific objectives were to determine the processing methods on:

a. the proximate composition of the flours;

b. the anti nutrient composition of the flours;

c. the raffinose, stachyose, hydrogen cyanide and haemagglutinin contents of the

flour; and

d. the organoleptic characteristics of their gruels

1.4 Significance of the study

The study would provide information on the nutrient, anti-nutrient, haemagglutinins,

hydrogen cyanide, raffinose and stachyose composition of African yam bean (AYB) flour.
 5

The gruels made from AYB flours would be a form of dietary diversification, which will

enhance AYB food use and contribute to ensuring food security and sustainability in Nigeria. It

may also stimulate local production and create employment for rural population. The study

would serve as baseline information for researchers in this area.

 6

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Legumes in human nutrition.

According to National Academy of Science (NAS) (1997), a legume is a simple dry

fruit that develops from simple carpel and usually opens along a seam on two sides. Grain

legumes are plants belonging to the legume family with papilionaceous flowers and pods

containing seeds. Most legumes do not need industrial fertilizers this is because of their natural

symbiosis with Rhizobium which provides them with organic proteins made directly from

atmospheric nitrogen (NAS, 1997). Grain legumes are cultivated primarily for their seeds

which are rich in energy and protein. Legumes are seeds that grow in pods; they are high in

fibre, low in fat and a good source of protein. Beans, lentils, peas, soybeans, and peanuts are all

examples of some major common legumes. Lesser known legumes are African yam beans,

baobab among others (Obiakor & Nwanekezi, 2008). A legume is a simple dry fruit that

develops from a simple carpel and usually opens on two sides. The seeds are hard and dried

and cannot be eaten unless prepared in the kitchen. For quite some time, legumes were

considered a rather lowly food; their food is increasing with recent discoveries concerning their

many nutritional and health properties (Pamplona-Roger, 2006).

Crosby (2009) stated that the plants show great diversity in both vegetative and floral

form; woody, perennial species predominate, but numerous herbaceous forms and even a few

aquatics also occur. The fruit is the feature by which the family is best characterized.

Technically known as a legume, it is a single-chambered, flattened seedpod with two sutures. It

usually splits open along the two sutures, as in the common pea. The seeds are attached along

one of the sutures. The legume may be indehiscent (not splitting), as in the peanut, which

matures underground; or explosively dehiscent, as in broom or lupine. It also may range from

only a few millimeters long to more than 30 cm (more than 12 in) and may be single or many

seeded and brightly or dully colored.

 7

According to Crosby (2009) the family of legume is divided into three closely related

subfamilies, which are often treated as three separate families. One subfamily is mostly

herbaceous and is characterized by simple leaves and highly irregular flowers with ten stamens

in two clusters. About 12,000 species exist, including such plants as peas, beans, peanuts, and

soybeans; clover and alfalfa; and sweet pea, broom, and lupine. The second subfamily contains

mostly trees and shrubs and is characterized by bipinnately compound (doubly branching)

leaves and regular (radially symmetrical) flowers with ten or more stamens extending beyond

the petals. This subfamily contains about 3,000 species and includes acacias and mimosas. The

third subfamily is also mostly woody, but with leaves pinnately compound, and slightly too

highly irregular flowers with ten stamens in one cluster. This subfamily contains about 3,000

species and includes such plants as brazil wood, carob, honey locust, Judas tree, logwood, and

tamar (Crosby, 2009).

2.2 Functional properties of legumes.

Legumes contains large group of non nutritive compounds known as photochemical

that are biologically active in the body, and they act as antioxidants, these photochemical were

earlier thought to be harmful to the body, but, with the recent research research, they were

discovered to have healing and protective properties. This phytochemicals fall in the class of

phenolic acids, saponins, isoflavones, phytates among others (Gropper, Smith, & Groff, 2005).

Legumes are low in fat and carbohydrate and high in protein, they can be used as substitute for

animal protein, they are equally rich in magnesium, iron, copper and folic acid. Vitamins such

as B1 B2 B6 niacin, folates have been associated with legumes; the iron in legumes is two to

three times more than that found in meat though not absorbed readily, and vitamin C from

other foods in the meals of legumes increases the absorption to levels similar to heme iron in

meat (Pamplona-Roger, 2006; Gropper, Smith, & Groff, 2005).

Pamplona-Roger (2006) reported that 15% to 30% of legumes’ dry weight is fibre and

this amount is superior to whole grains. It has been documented that they contain 2-3 times the
 8

protein of cereal grains and no other plant food is as rich in protein as legumes in their natural

state (National academy of Science, 1997, Pamplona-Roger, 2006).

2.3 Legumes in diet-related non communicable disease

Leguminous seeds have some anti-nutrients which contribute to the ability to lower

blood glucose level and such anti- nutrient includes enzyme inhibitors such as phytates and

possibly lectins, this has shown to produce hypoglycemic response in humans and rats

(Onyechi, 2009). Studies have shown that legumes prevent arterial hypertension and lowers

blood pressure because of their high levels of potassium and low sodium (Pamplona-Roger,

2006, Sacks et al., 2001).

Legumes combat damage that low density lipoprotein (LDL) cholesterol in the blood

can cause. It also have an advantage of low risk of heart disease and slow down the absorption

of carbohydrates. Soluble fibres in bean wards off the ups and downs in blood sugar levels

therefore are good for diabetic patients. More so, soluble and insoluble fibres keep constipation

at bay. Legumes have complex carbohydrates which are good in providing energy to the brain

and muscles, have low glycemic index, the fibre content lowers cholesterol levels, reduce the

risk of cancer and aids digestion. Water soluble non-starch polysaccharides (NSP) that have

viscous properties occur mostly in legumes. They have benefit in the prevention/management

of diabetes and cardiovascular diseases (Onyechi et al., 1998; Enwere, 1998). Whole grain

consumption was reported to be significantly and inversely associated with the

development/control of diabetes (Jenkins, et al., 2000)

It contains flavonoids which act as female hormone estrogen. Phytoestrogen, is the

estrogen like production from plants that are good for menopausal women.

2.4 Bean as a legume.

According to Microsoft Encarta (2009) most bean belong to the subfamily

Papilionoideae of the family Fabaceae and bean is common name widely applied to many

plants of the legume family. The seeds and pods of these plants are used for food and forage.
 9

The seeds themselves are also called beans and are valuable as food because of their high

protein content. The term bean is also applied to plants of other families, such as the Indian

bean, which is a North American species, and the sacred bean, or Indian lotus. The seeds or

fruits of certain other plants, such as the coffee tree and the castor-oil plant are also called

beans.

2.5 African yam bean (AYB) (Sphenostylis stenocarpa)

African yam bean is known and called different names by different tribes in Nigeria,

some of the names are Azama, Ijiriji, Azam, and Uzaaki in Igbo; Girigiri in Hausa; Akpaka in

Delta and Nsama in Ibibio. Other names are Okpodudu, Ahaja, Nzamiri, Odudu and Sese.in

Igala. In some parts of Ghana, it is called Kulege or Kutreku. AYB belong to the family:

Fabaceae (alt. Leguminosae) subfamily: Faboideae tribe: Phaseoleae subtribe: Phaseolinae.,

also placed in: Papilionaceae. It is also called yam pea in English and it is usually cultivated in

the following African regions Northeast Tropical Africa: Chad; Ethiopia East Tropical Africa:

Kenya; Tanzania; Uganda, West-Central Tropical Africa: Burundi; Central Africa Republic;

Zaire West Tropical Africa: Cote D'Ivoire; Ghana; Guinea; Mali; Niger; Nigeria; Togo

South Tropical Africa: Angola; Malawi; Zambia; Zimbabwe (USDA, 2007).

AYB is grown both for its edible seeds and its tubers (Klu et al., 2001). The seeds are

mostly used in some regions. It is a vigorous vine which twines and climbs to heights of about

3m and requires staking, with its prolific spattering of large flowers which may be pink,

purple, or greenish with white, making it an attractive ornamental (NAS, 1979). The slightly

woody pod which contains 20 to 30 seeds is up to 30cm long and mature within 170 days (Klu

et al., 2001). The seeds of AYB vary in sizes and shapes. The seed coat has a range of colours

from pale white to black with spotted or mottled grey, cream and brown in between. In

Nigeria, it is grown mostly in the northern part where it is grown mainly for its seed (Alozie, et

al., 2009).
 10

2.5.1 Nutritional/ chemical composition and organoleptic attributes of AYB

Roasted AYB bread according to Onyechi and Nwachi (2008) has more acceptable

colour than bread made from raw bean. Two legumes, AYB (Sphenostylis stenocarpa) and

cowpeas (Vigna unguiculata), were processed into akara, moimoi and porridge then analyzed

along with the raw samples for chemical, functional and sensory properties. Results showed

that all the samples (raw or processed) had similar and high crude protein content with an

average of 20.7%. The cowpea and AYB porridges had the highest (22.9%) and the lowest

(19.9%) crude protein, respectively. This showed that the two legumes are very good sources

of protein. Moimoi made from African yam beans had lower gelation capacity (19.4 w/v),

higher water absorption capacity (68.0 ml/g), and lower oil absorption capacity (35.9 ml/g)

than that made from cowpeas with 23.6 w/v, 54.0 ml/g and 41.0 ml/g, respectively. Similarly,

akara made from African yam beans had a higher gelatin capacity of 34.5 w/v, water

absorption capacity of 71.0 ml/g and a lower oil absorption capacity of 60.0 ml/g compared to

that made from cowpeas (30.6 w/v, 57.0 ml/g and 62.02 ml/g, respectively). Sensory

evaluation showed that moimoi and akara made from African yam beans were not significantly

different (p>0.05) in colour, flavour, texture and overall acceptability as compared to those

made from cowpeas indicating that AYB could be very useful in the preparation of moimoi

and akara. In general, processing into akara and moimoi improved the oil absorption capacity

of the AYB. The improved capacity to bind fat would be useful in ground meat formulations

such as sausage in addition to the usefulness in making moimoi and akara (Achinewhu & Akah

2003).

According to Eneche (2006), African yam bean though deficient in methionine and

cystine, has a high protein content of 21.6% and high in lysine. AYB can also be utilized as

complementary protein in our carbohydrate based foods to enhance their demand and improve

their quality. Incorporation of AYB flour into wheat based products greatly reduces the cost of
 11

importation of wheat and increases the utilization of this lesser known legume as well as the

protein content of our carbohydrate wheat-based foods.

A moimoi-like dish, similar to the very popular steamed cowpea dish was produced

from the AYB. The AYB moimoi was compared with cowpea and soybean moimoi by a panel

of eight (8) judges. The samples were compared for taste, colour, aroma texture and overall

acceptability. The texture, aroma and overall acceptability of cowpea moimoi ranked highest

followed by AYB moimoi and lastly soybean moimoi. Colour wise, AYB moimoi was

preferred over cowpea moimoi followed by soybean moimoi. Texture wise, AYB and cowpea

were scored equally followed by soybean moimoi. Proximate analysis of the product revealed

total carbohydrate content was 40.8%, crude protein was 18.4%, ash was 7.1%, crude fibre was

8.3% and crude fat was 25.4% (Peterside, Dosumu & Njoku, 2002).

Proximate analysis of AYB according to Onyechi, et al., (2008) showed that it contains

21.2g of protein, 1.9g fat, 3.5g ash, and 6.05g dietary fibre, 46.0g of sugars and 52.1g of total

carbohydrates. Obiakor (2008), stated that AYB has a high lysine content, the crude protein

vary from 21-29% of which lysine comprises of up to 8% of the protein, 50% of carbohydrates

and 5-6% of fibre. Evans and Bouttler (1974) stated that amino acid of AYB analysis indicate

that the lysine content is equal to or higher than that of soybean while most of the other

essential amino acids corresponds to the WHO/FAO recommendation.

The raw seeds contain on the average 21.29% crude protein, with mean cystine value of

1.28 implicating it as the limiting amino acid in AYB, however, the other essential amino acids

were present in higher concentrations when compared to other legumes (Uguru et al., 2001).
 12

Table 1: Proximate (% dry matter) and mineral (mg/100g dry matter) composition of
cream, brown and brown spotted (speckled) raw AYB seeds

Cream Brown Brown spotted(speckled)

Dry matter 86.38 88.97 85.88
Protein 21.1 21.2 21.5
Ash 2.70 3.50 3.10
Fat 2.30 1.90 2.10
Dietary fibre 17.5 21.3 18.6
Sugar 5.90 46.0 48.1
Starch 50.5 46.0 48.1
Total CHO 56.4 52.1 53.8
Ca 41.0 61.0 36.3
Fe 5.08 4.37 4.64
Zn 2.44 3.02 2.44
P 267 289 308
K 1430 1490 1512
Na 3.02 3.58 1.62

Source: Ene-Obong and Carnovale (1992).

 13

Table 2: Amino acid distribution of AYB

Amino Acids Quantity (g/16gN)

Lysine 7.40
Histidine 4.08
Arginine 5.28
Aspartic acid 11.64
Threonine 4.14
Serine 5.80
Glutamic acid 15.20
Proline 4.71
Glycine 4.54
Alanine 4.60
Cystine 1.72
Valine 5.43
Methionine 1.17
Isoleucine 4.44
Leucine 7.59
Tyrosine 4.02
Phenylalanine 5.92
Source: Ene-Obong and Carnovale, (1992).

Table 3: Antinutrient composition of 3 varieties of African yam bean (raw seed

characterization).

Antinutrient Quantify
α-amylase inhibitor (6-13) ug-1
Saponin (2-4) mg/kg
Trypsin inhibitor (0.7-3.0) TIU/mg
Total oxalate (21-35) mg/100g
Soluble oxalate (3-6) mg/100g
Tannins (0.9-20) mg/g
Phytic acid (4.5-7.3) mg/g
α-galactosides (2.3-3.4)g/100g
cyanogenic glucoside 225mg /kg
Source: Betsche, Azeke, Fretzdorff, & Buening-Pfaue (2007)
 14

2.5.2 Economic importance/ uses of AYB

It was reproted that storage roots of AYB can be processed into 'yam bean gari.' This is

similar to the current staple of West Africa, 'cassava gari,' granular flour. The bean could make

a significant contribution to the improvement of food support, especially where resources are

poor (The American Society of Agronomy, (ASA) (2007). In Ghana, the water drained after

boiling may be drunk by lactating mothers to increase their milk production (Klu, Amoatey,

Bansa and Kumaga, 2001). AYB seed can be used as ornaments for decoration (USDA, 2007).

2.5.4 Constraints in the use of AYB

AYB is one of the lesser-known legumes and has the peculiar problem associated with

legumes, It has high antinutrient content and hard to cook (HTC) phenomenon. It has beany

flavour which hinders its extensive utilization, coupled with the increase in the cost of

domestic fuel. African yam bean belong to the family of legumes. Legumes generally contain

anti nutritional factors and toxic compounds like trypsin inhibitor, tannins, phytates,

hemaglutinin and oligosaccharides. These inhibit the bioavailability of nutrients. AYB is also

reported to be associated with flatulence caused by flatulence inducing oligosaccharides

(Nnam, 1999., Ene-Obong & Obizoba, 1995).

Flatulence is the expulsion through the rectum of a mixture of gases that are byproducts

of the digestion process of mammals and other animals. The mixture of gases is known as

flatus and informally as a fart, or gas, and is expelled from the rectum in a process referred to

as "passing gas", "breaking wind" or "farting". Flatus is brought to the rectum by the same

peristaltic process, which causes faeces to descend from the large intestine. The noises

commonly associated with flatulence are caused by the vibration of the anal sphincter, and

occasionally by the closed buttocks. Intestinal gas is composed of varying quantities of

exogenous sources (air that is ingested through the nose and mouth) and endogenous sources

(gas produced within the digestive tract). The exogenous gases are swallowed when eating or

drinking or increased swallowing during times of excessive salivation. The endogenous gases
 15

are produced either as a by-product of digesting certain types of food, or of incomplete

digestion. Anything that causes food to be incompletely digested by the stomach and/or small

intestine may cause flatulence when the material arrives in the large intestine. Flatulence-

producing foods are typically high in certain polysaccharides, especially oligosaccharides. This

flatulence inducing oligosaccharides includes namely raffinose, stachyose and verbascose

which is due to the absence of α-galactosidase in humans are fermented anerobically to by

micro-organisms to produce carbon dioxide, hydrogen and methane (Suarez et al., 1999).

In beans, endogenous gases seem to arise from complex oligosaccharide

(carbohydrates) that are particularly resistant to digestion by mammals. These oligosaccharides

pass through the upper intestine largely unchanged. When they reach the lower intestine,

bacteria feed on them, producing copious amounts of flatus. Oligosaccharides when reduced

will have a phytochemical effect of growth promotion on bifidobacteria and because of that it

is said to promote the health of the colon, increase longevity and decrease colon cancer risk

(Hata, Yamamoto, Nakajima, 1991).

Hydrogen cyanide (HCN): Inhibits the activity of cytochrome C oxidase of the

mitochondrial electron transport chain thus preventing electron transfer from cytochrome a/a3

(electron carrier) to molecular oxygen (the final electron acceptor of the respiratory chain). It

may do this by combining with iron, (the catalytic group of the enzyme), thus eliminating the

active group involved in electron transfer to molecular oxygen hindering cellular oxidation and

the supply of energy to the cell. Heat treatment, thus, reduces the risk of dietary exposure of

consumers of AYB based diet to cyanide poisoning (Onyeike, & Omubo-dede, 2002; Oke,

1967).

Hemagglutinin (Lectins): refers to a substance that causes red blood cells to

agglutinate. They are carbohydrate-binding proteins (Hudson, 1984). The occurrence of lectins

is widespread in the plant kingdom, though more abundant in legumes.

 16

2.5.4 The antinutrients in AYB

Antinutrients are natural or synthetic compounds that interfere with the absorption of

nutrients (Atwood et al., 2006). Nutrition studies focus on those antinutrients commonly found

in food sources and beverages. The poor digestitibilty of the nutrients (especially protein) has

been attributed to the presence of anti nutrients and this limits the nutritional potentials of such

food for both humans and animals (Liener, 1973). Proteins can also be antinutrients, such as

the trypsin inhibitors and lectins found in legumes (Tan, Gyllenhaal, Soejarto, 2006). These

enzyme inhibitors interfere with digestion.

Phytic acid

Phytic acid is present in many plant systems, constituting about 1 to 5% by weight of

many cereals and legumes. Concern about its presence in food arises from evidence that it

decreases the bioavailability of many essential minerals by interacting with multivalent cations

and/or proteins to form complexes that may be insoluble or otherwise unavailable under

physiologic conditions (Cheryan and Rackis, 1980). Phytic acid content can be reduced

through soaking or other forms of processing (Realfoodmedia, 2009).

Tannins

Polyphenols (tannins) are usually located in the pericarp and/or testa, especially on

pigment cultivars of legumes and millets. Tannin concentration is reported to be higher in

coloured seed coats with a range of 38-43mg/g and low in white coated beans (1.3mg/g).

However, values ranged from 3.8-5.9mg/g in the cotyledons (Elias, Fernandez & Bressani,

(1979).

Saponins

Saponins are glycosides composed of a lipid-soluble aglycone that consists of either a

sterol or more commonly, a triterpenoid structure attached to water-soluble sugar residues that

differ in their type and amount. The major sources of dietary saponins are legumes, and many

types of saponins can be present in the same bean (Messina, 1999). Saponins are very poorly
 17

absorbed. They can kill or inhibit cancer cells without killing normal cells (Rao, 1996). Most

saponins form insoluble complexes with 3-ß-hydroxysteroids and are known to interact with

and form large, mixed micelles with bile acids and cholesterol (Malinow, Marbin &

delaCastra, 1985).

Trysin inhibitors

Trypsins inhibitors when ingested by man in large quantity disrupt the digestive

process and may lead to undesirable physiological reactions (Booth, Robbins & Kibellin,

1960). They co-exist with anti α-amylases and are located mostly in the outer layer of the

cotyledon of legumes. Trypsin inhibitors from beans interfere with protein digestion and in

some species of animals do cause pancreatic enlargement and enhance chemically induced

pancreatic tumors (Grant, 1989). However, heat-treating dry beans generally reduces the

trypsin inhibitor content by 80–90% (Duarte-Rayas, 1992). Other processing methods like

soaking in water through leaching, fermentation and germination has been shown to also

reduce trypsin inhibitors (Nnam, 1997; Obiakor, 2008; Obizoba & Atti, 1991; Ene-Obong and

Obizoba, 1995).

Oxalates

Oxalate occurs widely in the plant kingdom, examples of foods containing oxalates are

black pepper, parsley, poppy seed, amaranth, spinach, chard, beets, cocoa, chocolate, most

nuts, most berries, fishtail palms, New Zealand spinach (Tetragonia tetragonioides) and beans.

Excess consumption of oxalates may result in kidney disease or even death due to oxalate

poisoning (Streitweiser & Heathcock, 1976). Oxalic acid can induce toxic as well as anti

nutritive effects. To humans, it can be acutely toxic. However, it would require massive doses

of 4 to 5 g to induce any toxic effect (Oke, 1969). The oxalic acid levels usually found in food,

however, are no cause for concern. Like phytic acid, oxalic acid reduces the availability of

essential bivalent cations. Oxalic acid is a strong acid and, with alkaline earth metal ions and
 18

other divalent metal ions, it forms salts that are hardly soluble in water. Calcium oxalate is

insoluble in water at neutral or alkaline pH, and dissolves easily in an acid medium. Oxalates

produce irritation in the mouth and thereby preventing the absorption of calcium and iron in

foods (Osisiogu, Uzor, Ugochukwu, 1974; Oke, 1969). A study by Dresbach (1980) showed

that oxalate toxicity on calcium metabolism acts by combing with serum calcium to form an

insoluble calcium oxalate complex bringing about reduction in serum calcium level and violent

muscular stimulation with convulsion and eventual collapse. The reduction of oxalate to a

physiologically tolerable level by processing may enhance cellular utilization of some nutrients

for metabolic activities of some enzymes. Oxalates functions as chelating agents and may

chelate many toxic metals such as mercury and lead. Unlike other chelating agents, oxalates

trap heavy metals in the tissues (Shaw, 2010).

2.5.5 AYB processing methods used

Roasting

Roasting is a traditional processing technique, it has the capacity to develop attractive

flavours in foods so treated. It also induces important functional properties, attributes that

should be compatible with nutritional value (Bressani, 1983).

Fermentation

Fermentation is one of the oldest and cheapest traditional processing methods used in

the home and industries to improve the nutritional quality of food and reduces anti nutrient and

toxic substances like phytic acid, polyphenols and oxalic acids, Hydrogen cyanide, raffinose

and stachyose among others to improve food use (Gibson, 2007; Obizoba & Egbuna, 1992;

Nnam, 1994; Mahungu, Yawaguchi, Almazar, Kahan, 1987; Obizoba & Atti, 1991, Ogunsua,

1980). It is the metabolic process in which carbohydrates are oxidized with the release of

energy. During fermentation, the microbial enzymes converts storage nutrient in foods to

readily utilizable form (Rajalakshimi & Ramakrishanan, 1977). Fermentation begins when a
 19

food rich in simple sugars, yeast, and water are combined and left at room temperature. During

the first stage, the yeast cells multiply, using the sugars for energy, and produce small amounts

of alcohol (Byrd-Bredbenner, Moe, Bestgetoor, Berning, 2007). Many foods which are

inedible are made edible in their unfermented form and this is brought about by the extensive

hydrolysis of the indigestible components and the removal of antinutritional factors by the

micro-organisms (Odunfa, 1983).

Reasons for fermentation

Some reasons why fermentation of legumes is used in the preparation of foods are:

(1) Legumes often contain substances that are undesirable, such as the trypsin, phytates

among others. The treatments of the beans (soaking or heating) in preparation for

fermentation or the enzymes produced by the microorganisms remove or destroy these

factors.

(2) Moist products spoil readily, however, after fermentation, some products will keep

without refrigeration for extended periods of time. This is especially true of the

fermented products that are high in salt.

(3) All the legume fermentations involve the action of proteolytic and lipolytic enzymes

with the result that the final products are more digestible.

(4) Almost invariably the final product has a changed flavor more acceptable to the

consumer.

(5) In many instances the microorganisms increase nutrients such as vitamins, including

riboflavin and vitamin B12 in some of the bacterial fermentations.

(6) Finally, fermentation may reduce energy requirements. Thus, a short cooking may be
the major energy input required while the microorganisms do the rest of the work by
using energy from the substrate.
Source: Hesseltine & Wang (1980).
In addition, fermentation equally extends shelf life and level of safety (Hesseltine &

Wang, 1980). It increases essential amino acids like methionine, improved palatability,
 20

increase non protein nitrogen (Reddy & Salunkhe, 1980). Proteolytic acid and amylolytic

enzymes from micro-organism in fermentation process enhance digestibility and nutritional

quality (Murato, Ikehata & Myamoto, 1967). It equally enhances flavour, aroma, texture,

keeping quality and improves nutritive values (Eka, 1980). Fermented corn, cowpea and AYB

are known to have higher nitrogen balance than its unfermented counterparts (Nnam, 1999).

2.6 Lime

Lime is a citrus fruit. The lime tree (Citrus aurantifolia) belongs to the Rue family

(Rutaceae). Limes are small, oval citrus fruits with porous and smooth skin. Lime colour

ranges from light to medium green, sometimes with a slight yellow cast. Limes and lemons

look similar, but limes are smaller and green (when ripe) lemons are yellow

(NewWorldencyclopedia, 2010). Its fruits like other citrus fruits are very rich in vitamins A, B

and C. They have a tangy flavour and are used in cooking, baking, pickling, soaking among

others. It also contains various other minerals and acids. The fruit juice is an efficacious

remedy in scurvy, anaemia, intestinal disorders, cough and cold, gastric troubles, constipation,

fevers, typhoid and high blood pressure. It is a tonic, rejuvenative and refresher

(onlinefamilydoctors, 2000).Citric acid treated AYB has been shown to have greater reduction

in anti nutritional factors like the cyanides (Azeke et al., 2007). Citric acid treated AYB was

also found to be bacteria and cyanogenic glucoside free.

 21

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Materials

Cream coloured African yam bean (AYB) seed was purchased from Oye Igbo-Eze and

lime was purchased from Ogige Nsukka both in Enugu State, Nigeria.

3.2 Methods

3.2.1 Preparation of AYB flour for gruel and chemical analysis

Fifty-two cups (6kg) of cream coloured AYB seeds were sorted, weighed and divided

into 4 equal portions of 1.5kg each. These portions were labeled 24h fermentation without lime

and roasting (SO1), 24h fermentation with lime and roasting (SL2), 48h fermentation with lime

and roasting (FL3) and Roasted only (RO4).

 24h fermentation without lime and roasting (F24): - The sorted AYB seeds were

washed and fermented in tap water in a ratio of 1:3(w/v) for 24h at room temperature

(25±28 0C). The water was changed every six hours. At the end of 24h fermentation, the

seeds were sundried for 72h and roasted till cracking sets in. The roasting was carried

out by placing the sauce pan on fire and allowing it to heat. The AYB seeds were

poured into the saucepan and wooden spoon was used to stir the AYB seeds to prevent

burning. After roasting, the seeds were allowed to cool and milled whole into fine flour

using laboratory mill.

 24h fermentation with lime and roasting (FL24): - The sorted AYB seeds were

washed and soaked in tap water containing 30 tablespoonful of freshly squeezed lime in

a ratio of 1:3(w/v) at room temperature (25±280C) for 24hours. The water was changed

every six hours with addition of the same quantity of lime. At the end of 24 hours

fermentation, the seeds were sundried for 72 hours and roasted till cracking sets in: The

AYB seeds were poured into the saucepan and wooden spoon was used to stir the AYB
 22

seeds to prevent burning. After roasting, the seeds were allowed to cool and milled

whole into fine flour using the same method above.

 48h fermentation with lime and roasting (FL48): - The sorted AYB seeds were

washed and fermented in lime water (30 table spoonful of lime) for 48h at room

temperature (25±280C) in a ratio of 1:3(w/v). At the end of 48 hours of fermentation,

the seeds were sundried for 72 hours and roasted in a hot sauce pan till cracking set in.

The AYB seeds were poured into the saucepan and wooden spoon was used to stir the

AYB seeds to prevent burning. After roasting, the seeds were allowed to cool and

milled whole into fine flour using laboratory mill.

 Roasted only (OR): - The AYB seeds were washed, drained and roasted in a hot

saucepan until it starts cracking. The roasting was as thus: the pan was put on fire to

heat and the AYB seeds were poured into the pan using wooden spoon to turn the seed

to prevent burning. After roasting, the seeds were allowed to cool and milled whole

into fine flour using laboratory mill.

 The samples were separately stored in airtight containers for chemical analysis and

preparation of gruel.
 23

AYB SEED

HAND SORTED

Washed Washed Washed Washed

Drained

fermented in tap H2O fermented in tap H20 Fermented in tap H2O

Roasted (24hrs) (1:3w/v) containing lime (24hrs) containing lime (48hrs)
(till cracking) (F24) (1:3w/v) (Fl24) (1:3w/v) (FL48)

Milled
Sundried (72h)

Whole Fine Flour

(OR)
Roasted (till cracking)

Milled

whole Fine flour

Fig. 1: Flow chart for the processing of AYB flour
 24

3.2.2 Preparation of gruel from AYB flours

Gruel was prepared from each of the AYB flour samples.

Ingredient for the gruel preparation:

Flour ------- 400g

Water------- 2.5 litres (10 cups)

Sugar------- 4 cubes

Method of preparation:

500ml water was used to reconstitute the flour

1. 2 litres of water was brought to boil.

2. The boiled water was gradually added to the reconstituted flour while stirring

continuously to avoid the formation of lumps.

3. The mixture was allowed to simmer for some minutes and stirred continuously till

cooked (approximately 5 minutes).

4. Sugar was added and stirred

5. It was served hot

3.3 Laboratory analysis

Four samples of milled AYB flour were subjected to chemical analysis.

3.3.1 Protein determination

Protein was determined by automated micro-Kjedahl method described by AOAC

(1995). One gramme of each sample was weighed into the micro- Kjeldahl flask and 20ml

concentrated H2SO4, 2g Na2SO4, 0.5 CUSO4 (as catalyst) and 0.1g, selenium was added in the

flask. The mixture was boiled on a digester until the black solution became clear after which it

was made up to 100ml with distilled water. About 5 ml samples were drawn from the solution

and subjected to steam, boric acid, blue methyl and red methyl. The end product was titrated

against 0.01N HCL. The percentage nitrogen was gotten from the formula:
 25

% Nitrogen = Titre x N x DF x Nmw x 100

Weight of samples in mg

Titre = Final burette reading -initial burette reading

N = Normality of acid
DF = Dilution factor
NMN =Molecular weight of nitrogen.
Percentage protein = % Nitrogen x 6.25 (conversion factor).

3.3.2 Fat determination

The fat content of the sample was determined by Soxhlet extraction method (AOAC,

1995). Extraction flask was weighed; two grammes of each sample were weighed into a filter

paper and introduced into the extraction thimble. The thimble was placed into the soxhlet

extractor, some quantity of petroleum ether was placed into the flask and connected to the

soxhlet apparatus. The extraction lasted for about 6 hours at 40-60 0C after which the solvent

(petroleum ether) was recovered leaving only the extract in the flask. The extract was dried at

100 0C to expel the remaining solvent, then cooled in the dessicator and weighed.

% fat = (weight of flask + oil) – (weight of flask) x 100

Weight of sample

3.3.3 Ash determination

The ash content was determined using the official method of the AOAC (1995). Two

grammes of each sample were weighed into a weighed crucible and incinerated in a muffle

furnance at 6000C for about 6 hours. The crucible was removed and cooled in a desiccator and

reweighed.

% Ash = (Weight of crucible + Ash) – (Weight of crucible) x 100%

Weight of sample

3.3.4 Crude fibre determination

The method of Joslyn (1970) was used. Two grammes of defatted sample were

hydrolyzed in a beaker with 200ml of 1.25% H2S04 for 30 minutes, and then filtered under

suction, washed with hot distilled water and boiled again for another 30 minutes with 200ml of
 26

1.25%NaOH. The digested samples were washed with 1%HCLto neutralize the NaOH for

several times with hot distilled water. The residue collected was put into a weighed crucible

and dried at 100oC for 2 hours in an air oven. It was cooled and weighed. The ash was cooled

and weighed. The % crude fibre was calculated using the expression:

% crude fibre = loss in weight after ignition x100

Weight of sample

ie Crude fibre = weight after drying – weight after ignition x 100

weight of sample

3.3.5 Carbohydrate determination

This was determined by difference. The determined percentages of protein, fat, crude

fibre and moisture were summed up and subtracted from 100%.

3.3.6 Phytate determination

Phytate was determined by a simple and rapid colourimetric method by Eskin and

Latta, (1980). The equipment used were Anion exchange column, spectrophotometre,

centrifuge,volumetric flask and Vortex mixer. The reagents used were HCL, 2.4% HCL

(0.65N), Wade reagent: 0.03% FeCl3.6H2O and 0.3% sulfosalicylic acid) in distilled water

NaCl, 0.1M, 0.7M NaCl

Procedure

Five grammes (5g) of milled sample were weighed into a 250 ml conical flask, 100ml

of 2.45HCL was added and extracted for 1 hour at room temperature 25 0C±280c and

centrifuged. Supernatant was decanted. 1ml of 2.4% extract supernatant was diluted to 25 ml

with distilled water. Ten milliliters (10mls) of diluted sample was passed through the AG1-X8

chloride anion exchange column (0.5 g). Phytate was eluted with 0.7M NaCl. 3ml of 0.7M

eluent fraction was pipetted into 15 ml conical test tubes, and mixed on a vortex mixer for 5

seconds, and centrifuged for 10 minutes. Absorbance of supernatant was read at 500 nm using

water to zero the spectrophotometer

 27

Preparation of standard curve

Series of sodium phytate dilutions were made from 5-40 µg phytate in distilled water.

Three millimetres (3ml) of solution was pipetted into 115ml. One millimetre (1ml) of Wade

reagent was added within 30 minutes of elution. It was mixed on a vortex mixer for 5 seconds

and centrifuged for 10 minutes. Absorbance of supernatant was read at 500 nm using water to

zero the spectrophotometer

Phytate content was estimated from the standard curve.

3.3.7 Tannins

Tannins was determined using Joslyn, (1970) method. The reagents used were

methanol, sodium carbonate; 350g NaCO3 dissolved in 1 Litre of distilled water at 70-80 0C

cooled and filtered through glass wool. Tannic acid (100ppm) (0.01g tannic acid was weighed

and dissolved in 100ml of distilled water). Sodium tungstate, orthophosphoric acid,

phosphomolybdic acid, folin-Denis reagent: (50g of Sodium tungstate, 10g of

phosphomolybdic acid and 25ml of phosphoric acid was added to 375ml distilled water and

reflux for 2h, and allowed to cool and made up to 500ml mark.

Preparation of standard curve

1. 0, 0.05, 0.1,0.2, 0.3, 0.4 and 0.5ml of tannic acid standard solution was pipetted into test

tubes and made up to 5ml by adding 5, 4.95, 4.9, 4.8, 4.7, 4.6 and 4.5ml of distilled water

(These corresponds to concentrations of 0, 1, 2, 4, 6, 8 and10 ppm.)

2. 0.3ml Folin-Denis reagent was added

3. 0.6ml of Na2CO3 solution was added

4. The solution was allowed to stand for 25-30 mins. The absorbance of blue color was read

at 760nm.

Analysis of sample

One hundred miligrammes (0.01g) of sample were weighed; 20ml cold (4 oC) methanol

was added. It was vortexes and centrifuged at 3,000rpm for 20minutes.

 28

1. An aliquot of 0.01 to 5ml of supernatant was taken for assay.

2. % Tannins was calculated from standard curve as follows:

%tannins = (A-I) xVx100xD. F

BxWx106

A= Absorbance of sample.
I= Intercept
V= Total volume of extract
B= Slope of standard curve
W= Weight of sample

3.3.8 Determination of trypsin inhibitors

Trypsin inhibitor was determined using Kakade, Racis, Mcchee, & Puski (1974)

method. The reagents were prepared by dissolving 1.21g of hydroxymethyl amino methane and

0.59g of CaCl2 H20 in 180 ml of distilled water in Tris-buffer and BAPA solution. The pH was

adjusted to 8.2 with 1N HCl and put in water bath for 2 hours at 37 oC 0.08g of BAPA was

weighed and dissolved in 2ml of dimethisulfuroxide and added to the already prewarned

trisbuffer in a measuring cylinder and adjusted to 200 ml with distilled water. It was returned

to water bath for another 1 hour. Trypsin solution was prepared by weighing 0.0040g of trypsin

into a 250ml volumetric flask and added 200 ml of .001N HCl. and kept in the refrigerator

before and after use. Thirty grammes (30g) of glacial acetic acid were weighed and dissolved

in 70 ml of distilled water.(30% glacial acetic acid), 1 ml of 1N NaOH was used in 100 ml of

distilled water . One normal hydrochloric acid (IN HCl) was used in 100 ml of distilled water.

Procedure

One grammes (1g) of sample was weighed and 50 ml of 0.01N NaOH was added to

extract the sample. P H was adjusted between 8.4-10 0. 1N HCL was used and pH and IN

NaOH was reduced to increase the pH to required level. The sample was allowed to stay for 3

hours stirring at intervals to maintain the sample in suspension. 1ml of the extract was

withdrawn in 33ml of distilled water for dilution from the diluted extract; 2ml was taken and
 29

poured in 3 test-tube each. 2ml of each samples of trypsin solution was added to 2 test-tubes.

2ml of distilled water was withdrawn in 3 test-tubes and 2mls of trypsin solution was added to

2 test tubes. The samples in the test-tubes were returned to the water bath and allowed to warm

for 10 minutes. 5mls of BAPA was put to the entire test-tubes, vortex and warmed again for 10

minutes. 1ml of glacial acetic acid solution was added to all the test tubes and vortexed. 2ml of

trypsin solution was added to all the 3rd test tubes that do not contain trypsin solution initially.

The samples were filtered and the absorbance was read at 410nm using a spectrophometer.

Calculations:

T.I mg/g of sample = Abs of Standard - Abs of sample x dilution factor

0.019 x sample weight 1.000 x sample size (ml)

In 1g sample: T.I mg/g of sample = (Abs of standard –Abs sample) X dilution factor
19

Therefore = (Abs of standard –Abs of sample) X 50 X33

19

3.3. 9 Oxalic acid determination

Oxalate was determined by the method of Nwinuka et al. (2005). One hundred

milligrammes (0.1g) of the flour were extracted thrice by warming it at 40-50 degree

centigrade with constant stirring with magnetic stirrer for 1 hour with 20ml of 0.3 N HCL The

extract was diluted to 100ml with distilled water. 5ml of the extract was made alkaline with

1ml of 5 N NH4OH. This was made acidic with glacial acetic acid and phenolphthalein served

as an indicator (2 drops). 1 ml of 5% calcium chloride was added and the mixture was allowed

to stand for 3 hours, centrifuged using (IEC Centra GP8) at 1400 rpm for 15 minutes. The

supernatant was discarded and the precipitate was washed thrice with hot water, thorough

mixing and centrifuging each time. 0.2ml of 3 N H2SO4 was pipette to each test tube.

The precipitate was dissolved by warming in water at 70oC. for 30 mins. The content of

each test tube was titrated with freshly prepared 0.01 N Potassium permanganate solutions.

Titration was done at room temperature 29 oC until the colour of the solution become pink. The
 30

solution was allowed to stand until it became colourless. It was warmed at 70 degree C and

titrated until a pink color persisted for 30 seconds.

Calculations =oxalate content=W* 100/5

W= Mass of oxalate in 100ml

3.3.10 Determination of saponins

Saponins determination was carried out by Fenwick and Oakenfull, (1983) procedure. The

reagents used were reagent grade acetone, methanol, solution of concentrated n-buthanol-

methanol-ammonia (3.5:1:2.5), Standard solution of saponins purified in methanol, Solution of

sulphuric acid in methanol (100 ml per litre). The materials and equipment used were Soxhlet

extractor, Rotary evaporator, Densitometer with plotter,Planimeter, Dryer,Silica gel

chromatography plate (Kieselgel 60 F-254 Merck).

Method

The sample was finely ground and dried at constant weight. 40g were weighed and

placed in the Soxhlet reflux extractor with acetone for 24 hours. The solvent was changed for

methanol and extraction was continued for another 24 hours. The methanolic extract was

cooled and made to 250 ml with methanol.

At this point there was a modification to the method, proposed by Miriam Monforte

(CICY, Merida, Yucatan, Mexico). Instead of bringing the sample up to 250 ml as suggested in

the original method it was concentrated. In this, methanolic extract was transferred into a

rotary evaporator and concentrate until dry. The residue was concentrated again in a minimum

of methanol and transferred to a reweighed vial. The vial was weighed with the dry sample and

the weight of the residue was calculated.

Fine drops of a standard solution of saponins were placed on the chromatography

plates. The points of extract were placed so that each one is at the side of standard saponins

drops. The plates were revealed and the drops with aspersion and a solution of sulphuric acid
 31

in methanol, it heated at 110°C for 30 min. The intensity of the saponins stains was measured

with a densitometer and the peak areas were calculated on the plotter with a planimater. The

results were expressed as the relation (R) of the peak areas of the unknown sample in respect to

those of the standard. R2 was plotted against the volume of the drop of methanolized extract on

the plate. The downslope of the line ( was calculated by the least squares method), divided by

the gradient of a line derived from a master curve, to give the concentration of saponins in the

extract and thus the saponins content of the sample.

3.3.11 Hydrocyanic acid determination

Hydrocyanic acid was determined according to AOAC (1995) method. Using this

method, 10 g of the dried sample were allowed to soak in 30 ml of water for 4 hours before

steam distillation into 2.5% (w/v) NaOH. Eight milliliters of 6 N NH4OH and 2 ml of 5%

(w/v) potassium iodide were added to the distillate before titrating with 0.02 N AgNO3.

3.3.12 Determination of haemagglutinin by spectrometric method

Haemagglutinin was determined according to the method described by Onwuka (2005).

Two grammes (2g) of the sample were weighed into 40ml normal saline solution buffered at

PH 6.4 with 0.01m phosphate buffer solution. It was allowed to stand at room temperature for

30mins and centrifuged to obtain the extract. Half of a mililitre (0.5ml) of the extract was

diluted in a test tube, 1 ml of heparinized rabbit blood was poured. The blank was prepared by

adding 1ml of blood into a test tube and allowed to stand for 4h at room temperature. 1ml of

normal saline was added to all the test tubes and it was allowed to stand for 10min. after which

the absorbance was read at 620nm.

Haemagglutin unit/g= (b-a) x F

Where b = absorbance of the blank

F= experimental factor given by

F= (1/w x f/va) D
 32

Where

W= weight of sample

VF= total volume of extract

VA= volume of extract used in the assay

D= dilution factor

3.3.13 Raffinose and stachyose determination

Raffinose and stachyose (oligosaccharides) were determined by the method described

by Tanaka, Thanakul, Lee, and Chichester (1975). Five grams each of both raw and processed

flour were extracted with 50ml of 70% (v/v) aqueous ethanol and kept on an orbital shaker at

130 rpm for 13h. Extracts were further washed with 25ml of 70% (v/v) ethanol. The filtrates

obtained were then concentrated on a water bath. The concentrated sugar syrup was dissolved

in 5ml of distilled water. Separation of oligosaccharides was done by thin layer

chromatography (TLC). A 100g silica gel was dissolved in distilled water and stirred well until

the slurry was homogeneous. The TLC plates were washed, dried and cleaned with chloroform

to remove any grease from the plates. TLC plates were then coated with the slurry and air-

dried. Spotting of the sugar samples was done by using capillary tubes. Each sample was

spotted twice separately and dried using electronic hand drier. The plate were developed by

using a solvent system of n-propanol, ethyl acetate and distilled water (6:1:3), and dried. The

separated sugars’ colours were developed with iodine crystals. The separated spots were

compared with the standard sugar spots. The separated sugars that appeared were stachyose,

raffinose and sucrose. The stachyose and raffinose spots were scrapped, eluted in 2ml of

distilled water, kept over night and filtered throughWhatman No.1 filter paper. The filtrates

were then subjected to quantitative estimation. The eluted individual oligosaccharide was

estimated. One ml of the eluted and filtered sugar solution was treated with one ml of

concentrated HCl. The tubes were boiled in water bath for exactly 6 min. After cooling,

absorbances of the oligosaccharide contents were read using spectrophotometer 259 at 432nm.
 33

The absorbance values were used to calculate the concentration and mass of the

oligosaccharides. Average values of duplicate estimations were calculated and the

oligosaccharide contents expressed on dry weight basis.

3.4 Organoleptic evaluation

Gruel made from flour fermented without lime for 24h and roasted, soaked with lime

for 24h and roasted, fermented with lime for 48h and roasted and roasted only were displayed

for sensory evaluation using the preference test 9- point hedonic scale as described by

Ihekoronye and Ngoddy (1985). With this method, a 30 member panelist was selected to rate

the sample on a 9- point hedonic scale where 1 represents lowest and 9 represents the highest

for colour, consistency, flavour and general acceptability. The panelists were made up of

lecturers and post graduate students of the department of Home Science, Nutrition and

Dietetics, University of Nigeria, Nsukka. The gruels were presented in a food warmer and

coded. On arrival, the judges were served a hot coded AYB gruel using a soup plate. An

evaluation form was also given immediately to each of the judges. A glass of water was given

to rinse the mouth after each tasting; this was to avoid a carry over taste from preceding

samples.

3.4 Statistical analysis

Statistical Package for Social Sciences (SPSS) 2.0 computer software was used to

analyze the data. Means, standard deviation were calculated where appropriate. Analysis of

variance (ANOVA) was used to determine the treatment that was different from others in the

various parameters tested.

 34

CHAPTER FOUR

4.0 RESULTS

4.1 Proximate composition of the different AYB flour on dry weight basis

Table 4 presents the proximate composition of the different AYB flours on dry matter basis.

The results are determined at p<0.05.

4.1.1 Protein

The protein for each sample varied. The sample fermented in lime water for 24h and

roasted had the least protein content (20.96%). The sample that was fermented in tap water for

24h and roasted had the highest protein (33.08%). The sample that was only roasted had

24.19% protein and the sample fermented in lime water for 48h and roasted had 27.86%

protein.

4.1.2 Fat

The sample fermented in lime water for 48h and roasted had the least fat value

(3.68%). The sample that was fermented in lime water had comparable fat with the sample that

was only roasted (5.46% vs. 5.49%). The sample that was fermented in tap water for 24h had

4.90% fat.

TABLE 4: Effect of treatments on proximate composition of different AYB flour samples

on dry matter basis (%).

Samples Protein Fat Ash Crude fibre CHO

F24 33.08 a 4.90 b 3.11 a 4.28 b 54.63 c
FL24 20.96d 5.46 a 4.08 a 4.24 b 65.26 a
FL48 27.86b 3.68 c 3.84a 4.16 b 60.46b
OR 24.19c 5.49 a 3.13a 6.31 a 60.88b
LSD 3.23 0.56 1.02 2.03 4.38
*Means of three replications.
abcde
values with different superscripts on the same column are significantly different (P< 0.05).
F24- AYB fermented in tap water for 24hrs & roasted
Fl24- AYB fermented in lime water for 24hrs & roasted
FL48- AYB fermented in lime water for 48hrs &roasted
OR- AYB Only roasted (control)
 35

4.1.3 Ash

The ash values varied but not significant. The sample fermented in tap water for 24h

and roasted had the least ash value (3.11%). The sample that was fermented in lime water for

24h and roasted had the highest value for ash (4.08%). The samples that were only roasted and

that fermented in lime water for 48h had ash values of 3.13% and 3.84%, respectively.

4.1.4 Crude fibre

The sample that was only roasted had the highest fibre content (6.31%) which was

significantly higher than the other samples. The fibre for other samples was not significantly

different from one another. The sample that was fermented in lime water for 48h and roasted

had the lowest fibre content (4.16%). The samples that were fermented in lime water for 24h

and roasted and that fermented in tap water for 24h and roasted had fibre values of 4.24% and

4.28% , respectively.

4.1.5 Carbohydrate (CHO)

The sample that was fermented in lime water for 24h and roasted had the highest CHO

value (65.26%) which was significantly different from the other samples. The sample that was

fermented in tap water for 24h and roasted had the least CHO level (54.63%) which was

significantly different from the other samples. The samples that were fermented in lime water

for 48h and roasted and that which was only roasted had CHO values of 60.46% and 60.90%

CHO respectively. There was no significant difference between the values.

4.2 Effect of treatment on the anti-nutrient content of AYB flour samples

Table 5 presents the effect of treatments on the anti nutrient contents of AYB flour samples.

Results are reported at p<0.05

4.2.1 Phytate

The sample that was only roasted had the least phytate (2.68mg/g) level, which was

similar to the phytate value of the sample fermented with lime for 48h and roasted (2.73mg/g).

The sample that was fermented in lime water for 24h and roasted had the highest level of
 36

phytate (2.97mg/g), which was significantly different from the levels in the other samples. The

sample that was fermented in lime water for 24h and roasted had a phytate level of 2.81mg/g

which was significantly different from the other samples (p<0.05).

Table 5: Anti nutrient composition of AYB flours (mg/g)

Samples Phytate Tannins Saponins Oxalate Trypsin inhibitors

F24 2.81b 0.02c 0.01a 0.01 b 0.48 ab
FL24 2.97a 0.03ac 0.01a 0.01 b 0.51 ab
FL48 2.73c 0.04ab 0.01a 0.03a 0.53 a
OR 2.68d 0.03ac 0.01a 0.01 b 0.45 b
LSD 0.05 0.01 0.02 0.16 0.08
*Means of three replications.
abcde
values with different superscripts on the same column are significantly different (P< 0.05).
F24- AYB fermented in tap water for 24hrs & roasted
FL24- AYB fermented in lime water for 24hrs & roasted
FL48- AYB fermented in lime water for 48hrs &roasted
OR- AYB roasted only (control)

4.2.2 Tannins

The sample that was fermented in tap water for 24h and roasted had the least tannin

level (0.02mg/g), which differed from the sample that was fermented in lime water for 48h and

roasted. The sample fermented in lime water for 48h and roasted had the highest level of

tannins (0.04mg/g). The samples that were only roasted and the one fermented in lime water

for 24h and roasted had the comparables tannins (0.03mg/g).

4.2.3 Oxalates

The differences in the oxalate levels were not significant. The sample fermented in tap

water and roasted, the sample fermented in lime water and roasted and the sample that was

only roasted had the lowest oxalate level of 0.01mg/g. The sample that was fermented in lime

water for 48h and roasted had the highest oxalate level of 0.03mg/g.

4.2.4 Saponins

The saponins content of all the samples were comparable (0.01mg/g) and did not differ

(p>0.05).
 37

4.2.5 Trypsin inhibitor

The roasted sample had the lowest trypsin inhibitor (0.45mg/g), which was significantly

different from the sample fermented in lime water for 48h and roasted (0.53mg/g). The sample

fermented in lime water for 48h and roasted had the highest trypsin inhibitor. The samples

fermented in tap water for 24h and roasted and that fermented in lime water for 24h and

roasted had trypsin inhibitor levels of 0.48mg/g and 0.51mg/g, respectively.

4.3 Effect of treatment on the raffinose, stachyose, hydrogen cyanide and

haemagglutinin composition of AYB flours

Table 6 presents the effect of treatment on the raffinose, stachyose, haemagglutinins

and hydrogen cyanide composition of AYB flours. Results are reported at P<0.05

4.3.1 Raffinose

The raffinose values for all the samples varied. Raffinose level was highest in the

sample that was only roasted (9.22mg/100g). The sample that was fermented in tap water for

24h and roasted had the lowest raffinose (8.25mg/100g). The samples that were fermented in

lime water for 48h and roasted and the one fermented in lime water for 24h had a raffinose

level of 8.45mg/100g and 8.78mg/100g, respectively.

4.3.2 Stachyose

The sample fermented in lime water for 24h and roasted had the least stachyose

(6.76mg/100g), while the sample that was only roasted had the highest stachyose

(8.48mg/100g). The samples that were fermented in tap water for 24h and roasted and the

sample fermented in lime water for 48h and roasted had a stachyose level of 7.19mg/100g and

7.51mg/100g respectively.

4.3.3 Haemagglutinin

The values for haemagglutinin differed. The sample that was fermented in lime water

for 24h and roasted had the least haemagglutinin (4.87hu/100g). The sample that was
 38

fermented in tap water for 24h and roasted had the highest haemagglutinin (6.70hu/100g). The

samples that were only roasted and the one fermented in lime water for 48h and roasted had

5.30hu/100g and 5.80hu/100g, respectively.

4.3.4 Hydrogen cyanide (HCN)

The sample fermented in lime water for 24h and roasted had significantly lower

hydrogen cyanide (0.22mg/g) than other samples. The sample fermented in lime water for 48h

and roasted had a HCN level of 0.23mg/g, the sample that was fermented in tap water for 24h

and roasted had 0.24mn/g HCN level. HCN was highest in the sample that was only roasted

(0.28mg/g).

Table 6: Raffinose, stachyose, heamagglutinins and hydrogen cyanide composition of

AYB flours
Samples Raffinose Stachyose Haemagglutinins Hydrogen cyanide
(mg/100g) (mg/100g) mg/100g mg/g
F24 8.25d 7.19 b 6.70 b 0.24 b
FL24 8.78b 6.76 b 4.87 e 0.22 c
FL48 8.45c 7.51 b 5.80 c 0.23 b c
OR 9.22a 8.48 a 5.30 d 0.28 b
LSD 0.20 0.32 0.25 0.04
*Means of three replications.
abcde
values with different superscripts on the same column are significantly different (P< 0.05).
F24- AYB fermented in tap water for 24hrs & roasted
FL24- AYB fermented in lime water for 24hrs & roasted
FL48- AYB fermented in lime water for 48hrs &roasted
OR- AYB roasted only (control).

4.4 Effect of processing on the organoleptic properties of the gruels

Table 7 presents the organoleptic characteristics of AYB gruels.

4.4.1 Colour

The sample that was fermented in lime water for 48h and roasted had the highest

(p<0.05) for colour (6.53). The colour of the gruel made from the sample that was fermented in

lime water for 24h and roasted had the least score for colour. It was neither liked nor disliked

(4.83), the colour of the gruels made from the samples that were fermented in tap water for 24h

and roasted (5.87) and the sample that was roasted only (6.10) were liked slightly.
 39

4.4.2 Flavour

The sample fermented in lime water for 24h and roasted had the highest score (p<0.05)

for flavour, it was liked moderately (6.57).The sample that was only roasted and the sample

fermented in lime water for 48h and roasted were liked slightly and had scores of 5.67 and 6.00

respectively. The gruel made from the sample that was fermented in tap water for 24h and had

the least (p<0.05) rating for flavour (5.50), though it was liked slightly.

4.4.3 Consistency

The consistency of all the gruels was similar. The consistency of the gruel fermented in

lime water for 24h and roasted (6.17) and fermented in lime water for 48h (6.13) and roasted

were liked slightly, while the sample that was fermented in tap water for 24h (6.87) and roasted

only (6.97) were liked moderately with scores of 6.87 and 6.07, respectively.

4.4.4 Degree of acceptability

There were variations in the general acceptability of the gruels. The gruels made from

the sample that was fermented in lime water for 48h and roasted, fermented in tap water for

24h and roasted and fermented in lime water for 24h and roasted were neither liked nor

disliked. The roasted was liked slightly (6.03) which was the highest score for general

acceptability statue.

Table 7: Organoleptic characteristics of AYB gruels

Colour Flavour Consistency Degree of acceptability

a b a
F24 5.87 5.50 6.87 5.17 b
FL24 4.83 b 6.57 a 6.17 a 5.30 a
FL48 6.53 a 6.00 a 6.13 a 4.93 b
OR 6.10 a 5.67 b 6.97 a 6.03 a
LSD 1.04 0.33 0.70 0.13
*mean± standard deviations of 30 panelist response on a 9-point hedonic scale with 9 = like extremely to
1 = dislike extremely.
abcde
values with different superscripts on the same column are significantly different (P< 0.05).

F24- AYB soaked in tap water for 24hrs & roasted

FL24- AYB soaked in lime water for 24hrs & roasted
FL48- AYB fermented in lime water for 48hrs &roasted
OR- AYB roasted only
Key: organoleptic scores
1. Dislike extremely 6. Like slightly
2. Dislike very much 7. Like moderately
3. Dislike moderately 8. Like very much
4. Dislike slightly 9. Like extremely
5. Neither like or dislike
 40

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 Discussion

5.1.1 Effect of processing on proximate composition of AYB flours

Protein

The increases and decreases in protein when compared with the control (roasted only)

were solely attributed to treatments. The lower protein content for the sample fermented in

lime water for 24h could be due to leaching of protein in the treatment medium. This

observation is in conformity with the work of Wanjekece, Wakasa, & Mureithi (2003). The

high protein content of the sample fermented in tap water for 24h could be attributed to the

processing method used (Mugendi, Ngaji, Kuria, Mwasaru, Mureithi & Apostolides, 2010;

Bressani, 1983; Borhade, Kadam & Salunkhe, 1984). This observation was because, during

fermentation there was hydrolysis of protein enzyme tannin-complexes to release free amino

acids for new protein synthesis (Tongul, Nanson, & fields, 1981). Fermentation is equally

known to cause gradual increase in crude protein of AYB and cowpea flours (Nnam, 1997).

Fat

The lower fat content of the 48h lime fermented sample might be due to metabolic

activities of micro organism and length of fermentation (48h). Vander Riet, Wight, Ciller &

Datel (1987) observed that fat decreases as fermentation time increases. The reducd fat content

of the fermented sample implies better keeping quality due to less rancidity of the flour.

Crude fibre

The decrease in the fibre content of the fermented samples is in line with other studies

that reported decrease in crude fibre during fermentation (Eka, 1980; Achinewhu & Iscichei,

1990).
 41

Carbohydrate

The decrease in the carbohydrate content for the sample fermented for 24h in tap water

might be due to high utilization of energy by micro flora during fermentation as observed in

earlier studies (Obiakor & Nwanekezi; 2009, Nnam, 1995), the decrease might also be due to

the significant increase in the protein content.

5.1.2 Effect of treatments on the anti nutrient composition of AYB flours

Phytate

The high phytate level for the sample fermented for 48h in lime could be as a result of the lime

treatment. All the samples, including the control appeared to be safe. The low anti-nutrients

levels suggest that minerals otherwise chelated by phytate can be much more available.

Reports from an earlier study showed that low phytate levels of 3.01mg/g had significant

increase in calcium absorption (Heaney, Weaver & Fitzsimmons, 1991).

Tannins

The higher tannins in 48h lime fermented sample could be as a result of the addition of

lime. Recent research shows that the consumption of tannins within 0.05-0.2% (150-

200mg/100g) of a diet can be regarded as safe (Schiavone, et al., 2007). Therefore, the tannin

level observed in this study would not exert negative effect associated with tannins such as the

lowering of available protein by antagonistic competition, this can elicite protein deficiency syndrome.

Oxalate

The higher oxalate for the 48h lime fermented sample could be as a result of the

addition of lime. The levels of oxalates in all the samples were within safe level (4-5mg/g).

Pearson (1973) reported that lethal dose of oxalate is between 200mg/100g and 500mg/100g.

Oke (1969) reported that low levels of oxalates (4-5mg/g) is known to cause no irritation in the

mouth or interfere with iron or calcium absorption. Dresbach (1980) reported that the reduction
 42

of oxalate to a physiologically tolerable level by processing enhanced cellular utilization of

some nutrients for metabolic activities of some enzymes.

Trypsin inhibitors

The higher trypsin inhibitors observed in the 48h lime fermented sample could be as a

result of the addition of lime. However, the level of trypsin inhibitor observed from all the

treated samples could be regarded as low and might be safe for consumption as the nutrient

which trypsin inhibitors interfers with can be made more available. Trypsin inhibitors in the

samples may not cause pancreatic enlargement and enhance chemically induced pancreatic

tumors. Low levels of trypsin inhibitors (0.54mg/g) have been shown to have no damage to the

pancrease (Harwood, 1986).

5.1.3 The effect of processing on the raffinose and stachyose contents of AYB flours.

The greater reduction of raffinose in all the samples treated is of interest. This is

because raffinose is implicated in flatulence. The observation is similar to the reports of many

workers (Nnam, 1997; Nwibani, Nwinuka, Bene, Abbey & Ayalogu, 2009). Fermented milk

has been shown to have greater reduction in raffinose over stachyose (Nnam, 1997).The much

more reduction in raffinose due to lime treatment agrees with the results that acidic medium

fastens the reduction of oligosaccharide, especially raffinose sugar (Jean et al, 2004).

5.1.4 Effect of treatment on the haemagglutinin and hydrogen cyanide composition of

AYB flour samples.

Heamagglutinin

The much higher reduction of haemagglutinin in the 24h lime treated sample could be

due to the intermittent changing of water and lime treatment provided acidic medium which

lowered haemagglutinin on the sample (Puri, Booy, Doms, White & Blumenthal, 1990). The

combination of fermentation in lime water for 24h and roasting appears to be most effective in

reducing haemagglutinin in AYB.

 43

Hydrogen cyanide (HCN)

The reductions of HCN in all the samples were far beyond the 35mg/100g lethal value

(Oke, 1969). The much more reduction in lime treated samples agreed with previous result.

This result indicated that treated AYB in citric acid medium led to the reduction of cyanide in

AYB (Azeke, 2007). From this study, fermentation in lime water for 24h in combination with

roasting is the most effective way to reduce HCN in AYB.

5.1.5 Effect of treatment on the beany flavour and other organoleptic characteristics of
AYB gruel

Colour

The higher rating for colour in AYB gruel made from the sample that was fermented in

lime water for 48h and roasted might be associated with the brown colour due to these

treatments. This assumption is in line with previous study of Hesseltine & Wang (2009). They

reported that fermentation and roasting improves colour by imparting brown colour to the

product, this brown colouration could be enzymatic browning, caramelization of sugar or even

Millard reaction. Nnam (1997) reported that the colour of the fermented AYB milk was

slightly preferred to the unfermented milk. Another study by Onyechi and Nwachi (2008)

reported that roasting improves colour. The brown colour observed in this sample can also be

attributed to the higher tannin content of the sample. Tannins are known to be responsible for

the pigmentation and browning of foods (Friday, Uhegbu, Onwuchekwa, Iweala, & Kanu,

2009).

Flavour

The preference of flavour to the control was a confirmation of previous studies of the

importance of processing for improved usage of traditional foods (Nnam, 1997; Betsche et al.,

2005; Obizoba, & Egbuna, 1992; Nnam, 1994; Nnam, 2003). The higher preference for lime

treated gruels might be attributed to masking of beany flavour of AYB due to synergistic effect

of roasting and fermentation in lime water. Lime is known to improve flavour of grains such as
 44

legumes and cereals (OnlineFamilydoctors, 2000; NewWorldencyclopedia, 2010; Fisher and

Bender, 1975). Anecdotal reports and daily practice showed that lime was used successfully to

mask strong flavour associated with foods such as fresh sea fish; Kunun Gyda ( Drink made

from groundnut) usually used by the Hausas; in the bakery industries as flavouring agents and

in beverage industries among others. From the study, fermentation in lime water for 24h in

combination with roasting has shown to be a better way of improving the flavour of AYB.

5.2 Conclusion

This work has shown that adequate knowledge of processes to increase nutrients and

reduce anti nutrients and anti physiological factors would increase food usage of AYB. It also

shows that the increased nutrients and reduced anti-nutrients, anti physiological factors and

toxic substances in AYB were attributed to synergistic effects of food processing methods

adopted. The 24h fermentation in tap water in combination with roasting increased protein

much more than other methods. Fermentation in lime water for 24h decreased protein and

increased carbohydrates. Fermentation in lime water for 48h reduced fat for better shelf life.

The samples that were only roasted had better fibre content. The 48h fermentation in lime

water increased the antinutrients, though the values are still within the safe levels.

Fermentation in lime water for 24h had greater reduction in the haemagglutinins level of the

flour samples, generally, lime treated samples had greater reduction in the raffinose, stachyose

and HCN contents of the flours. The 24h fermentation in lime water in combination with

roasting had positive effect on the beany flavour better than in the other products.

5.3 Recommendation

It is recommended that the combinations of different techniques of processing are

important to produce (wholesome) flours free of beany flavour. Therapeutic studies of AYB

products are imperative. Detailed studies are required in the area of lime treatment of AYB

flours. Food industries are recommended to process AYB flours using these processing

methods, package it well and make it available for buyers.

 45

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 53

APPENDIX 1

HEDONIC SCORING FORM FOR EVALUATION OF AFRICAN YAM BEAN

GRUEL

Taste these samples and check how much you like or dislike each one on the hedonic scale by

giving a score corresponding to each description below. Please give a reason for this attitude.

An honest expression of your personal feeling will help us.

FLAVOUR COLOUR CONSISTENCY TEXTURE

F24
FL24
FL48
OR

KEY: Organoleptic Scores

10. Dislike extremely
11. Dislike very much
12. Dislike moderately
13. Dislike slightly
14. Neither like or dislike
15. Like slightly
16. Like moderately
17. Like very much
18. Like extremely

Reasons for your attitude for:

F24:------------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------
FL24-----------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------
FL48:----------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------
OR--------------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------
 54

APPENDIX II

GENERAL ACCEPTABILTY RATING OF THE SAMPLES

DEGREE OF ACCEPTABILITY SAMPLES

F24 FL24 FL48 OR

9 I WOULD EAT THIS AT EVERY

OPPORTUNITY

8 I Would eat this often

7 I would eat this occasionally

6 I would eat this when available

5 I would when there is no option

4 I don’t like this but would manage

3 I would hardly ever eat this again

2 I would eat this only if I were forced

1 No account would I eat this

 55

APPENDAGE III

Raw cream coloured AYB seeds

 56

Gruel from AYB flour samples

OR (control)