TITLE PAGE

PRODUCTION AND EVALUATION OF EXTRUDED SNACKS FROM COMPOSITE

FLOUR OF BAMBARA GROUNDNUT ( Voandzeia subterranea (L) Thoaur ),

HUNGRY RICE (Digitaria exilis Staph.) AND CARROT ( Daucus carota L.)

A THESIS SUBMITTED TO THE DEPARTMENT OF FOOD SCIENC E AND

TECHNOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMEN TS FOR THE

AWARD OF MASTER OF SCIENCE (M.Sc.) DEGREE OF THE UN IVERSITY OF

NIGERIA.

BY

OKAFOR, JANE NGOZI

PG/M.Sc./07/42659

DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY

UNIVERSITY OF NIGERIA, NSUKKA

FEBRUARY, 2010

ii

CERTIFICATION

OKAFOR , JANE NGOZI, a postgraduate student in the Department of Food Science

and Technology, Faculty of Agriculture, University of Nigeria, Nsukka, has satisfactorily

completed the requirements for the degree of Master of Science (M.Sc.) in Food

Science and Technology. The work embodied in this dissertation is original and has

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

University.

______________________ ________________

Dr. (Mrs.) J. C. Ani Date Supervisor

______________________ ________________

Dr. (Mrs.) J. C. Ani Date Head of Department

iii

ACKNOWLEDGEMENT

My profound gratitude goes to the Almighty God for His mercies and graces. I wish to

thank Dr. (Mrs) J.C. Ani for her supervisory roles and tireless efforts that aided the

successful completion of this work. My sincere appreciation goes to my husband Dr.

G.I. Okafor for his love, support and encouragement.

I am very grateful to the entire Lecturers of the Department of Food Science and

Technology, University of Nigeria, Nsukka for enriching me academically.

I wish to thank my friends and well-wishers, who in no small measure have helped and

encouraged me in the course of this research work.

May the Almighty God bless and enrich you all abundantly.

Jane Ngozi Okafor

iv

ABSTRACT

Cleaned Bambara groundnut seeds were divided into four lots. Each lot was separately

pretreated thus: germinated, roasted, germinated and roasted, and unprocessed which

served as control. Each sample was ground, sieved, and extruded using single screw

extruder. Consumer preference test was done by a taste panel of 50 people who rated

the products on the attributes of colour, taste, flavour and overall acceptability using a

9-point hedonic scale. The treatment (roasting) given on the most preferred product

was adopted in producing composite of bambara and “acha” flour, which was mixed

with graded levels of carrot and other ingredients, and extruded. The product samples

were subjected to analyses for chemical composition, residual anti-nutrients, physico-

chemical and sensory properties using standard methods. The samples were stored for

six months under ambient conditions (28±2ºC) and analysed at 2 months interval for

moisture, texture, provitamin A (β-carotene) and sensory properties. Extruded snacks

from the composite of bambara groundnut, hungry rice and carrot had high protein (15-

16%), β-carotene (180-550.13mg/100g retinol) and minerals (iron and zinc) contents.

Inclusion of carrot to the composite increased (p≤0.05) the β-carotene content of the

product, when compared with the control. There were no significant differences

(p>0.05) between the sensory qualities of the control and products with 5% to 15%

carrot. Extrusion cooking significantly (p<0.05) reduced moisture content and brought

about concentration of other proximate components. It also significantly (p≤0.05)

reduced phytate from 91.01-81.11mg/100g to 36.75-30.58mg/100g, tannin from

0.16mg-0.26/100g to 0.06-0.09mg/100g. Trypsin inhibitor and haemagglutinin activities

were reduced from 6.81-8.32mg/100g and 4.01-6.50Hu/mg protein, respectively, to

undetectable levels. Extrusion cooking improved protein digestibility of the snacks. β-

carotene and minerals were not significantly (p>0.05) affected by the extrusion cooking,

while there was a significant reduction (p<0.05) of vitamin C from 6.21-8.96mg/100g to

2.51-4.05mg/100g in the extruded snacks. Significant (p<0.05) reductions were

observed in vitamin B1(40-50%), B2(15-24%), B3(15-24%) and B6(25-30%) content of

the extruded snacks. Storage for six months did not adversely influence the sensory

characteristics of the developed snacks. It is evident from the composition of the

developed products that protein-energy malnutrition and micronutrient deficiency

problems can be averted through dietary diversification and extrusion cooking

technology.

v

TABLE OF CONTENTS

Title Page - - - - - - - - - - i

Certification - - - - - - - - - ii

Acknowledgement - - - - - - - - - iii

Abstract - - - - - - - - - - iv

Table of Contents - - - - - - - - - v

Chapter One

1.0 Introduction - - - - - - - - - 1

1.1 Statement of the Problem - - - - - - - 2

1.2 Significance of the Study - - - - - - - 3

1.3 Objectives of the Study - - - - - - - 3

Chapter Two

2.0 Literature Review - - - - - - - - 4

2.1 Extruded Snacks - - - - - - - - 4

2.2 Legumes - - - - - - - - - 5

2.2.1 Bambara groundnut - - - - - - - 7

2.2.2 Processing of legumes - - - - - - 7

2.2.3 Milling and grinding - - - - - - - 7

2.2.4 Soaking, boiling and steaming - - - - - 8

2.2.5 Germination - - - - - - - - 8

2.2.6 Roasting - - - - - - - 9

2.2.7 Preparation of protein concentrate - - - - 9

2.2.8 Canning of legumes - - - - - - 10

2.2.9 Quick cooking legumes - - - - - - 10

2.2.10 Nutritional importance of legumes - - - - 10

2.2.11 Antinutritional constituents of legumes - - - - 12

2.2.11.1 Protease (Trypsin) inhibitor - - - - 12

2.2.11.2 Phytates - - - - - - - 13

2.2.11.3 Polyphenols (Tannins) - - - - - 13

2.2.11.4 Cyanogens - - - - - - 14

vi

2.2.11.5 Lectins - - - - - - - 14

2.2.11.6 Effect of processing on legume food quality - - 15

2.2.11.7 Effect of processing methods on anti-nutrients - 17

2.3 Extrusion cooking and its effect on food quality 18

2.3.1 Application of extrusion cooking 19

2.3.2 Advantages of extrusion cooking 19

2.3.3 Effect of extrusion cooking on protein quality 19

2.3.4 Effect of extrusion cooking on the anti-nutrients 20

2.3.5 Effect of extrusion cooking on amino acids 21

2.3.6 Effect of extrusion cooking on Maillard reaction 22

2.3.7 Effect of extrusion cooking on carbohydrates 23

2.3.8 Effect of extrusion cooking on vitamins 24

2.3.9 Effect of extrusion cooking on minerals 25

Chapter Three

3.0 Materials and Methods - - - - - - 26

3.1 Materials - - - - - - - - 26

3.2 Methods - - - - - - - - 26

3.2.1 Preparation of samples - - - - - 26

3.4.0 Analysis of Raw Material and Product - - - - 31

3.4.1 Proximate composition - - - - - 31

3.4.2.0 Mineral content analysis - - - - - 33

3.4.2.1 Determination of phosphorus - - - - 33

3.4.2.2 Determination of Iron - - - - - 34

3.4.2.3 Determination of calcium - - - - - 35

3.4.2.4 Determination of potassium - - - - 35

3.4.2.5 Determination of manganese - - - - 35

3.4.2.6 Determination of copper - - - - - 36

3.4.2.7 Determination of magnesium - - - - 36

3.4.3 In vitro protein digestibility - - - - - 36

3.4.4 Functional properties - - - - - 37

3.4.5 Determination of anti-nutritional factors - - - 37

3.4.6.0 Determination of vitamins - - - - - 40

3.4.6.1 β-carotene and Vitamin A - - - - - 40

vii

3.4.6.2 Vitamin B1 - - - - - - - 41

3.4.5.3 Vitamin B2 - - - - - - - 41

3.4.6.4 Niacin - - - - - - - 42

3.4.6.5 Vitamin C - - - - - - - 42

3.4.7.0 Experimental design/data analysis - - - 43

Chapter Four

4.0 Results and Discussion - - - - - - 44

4.1 Effect of Processing Treatment on the Proximate Composition of

Bambara Groundnut - - - - - - 44

4.2 Effect of Processing Method of Some Anti-Nutritional Factors 44

4.3 Effect of Processing Methods on the Functional Properties of

Bambara Groundnut - - - - - - 46

4.4 Effect of Treatment Method on Consumer Acceptability of

Extruded Snacks 47

4.5 Proximate Composition of Hungry Rice “acha” and Fresh Carrot 48

4.6 Effect of Extrusion on the Proximate Composition of Bambara/

Acha Blends Fortified with Carrot - - - - - 49

4.7 Sensory Qualities of Extruded Snacks - - - - 51

4.8 Effect of Extrusion on the Residual Anti-nutrients - - 51

4.9 Effect of Extrusion on in-vitro protein Digestibility - - 52

4.10 Effect of Extrusion on the Mineral Content of the Bambara

groundnut/Acha Blends Fortified with Carrot - - - 53

4.11 Effect of Extrusion on the Vitamin Content of the Snacks - 56

4.12 Effect of Storage on the Texture of the Extrudates - - 58

4.13 Effect of Storage on the Sensory Qualities of the Snacks - 59

4.14 Effect of Storage on the Vitamin Content of the Snacks - 60

Chapter Five

5.0 Conclusion and Recommendations - - - - 62

5.1 Conclusion - - - - - - - - 62

5.2 Recommendations - - - - - - - 62

References - - - - - - - - - 63

viii

LIST OF TABLES

Table 1: Trend in Total Distribution of Major Food Crops in Nigeria (‘000 tonnes) 6

Table 2: Energy and selected nutrients in some legumes - - - 11

Table 3: Essential Amino Acid Composition of Selected Legumes (g/16gn) - 11

Table 4: Effects of Antinutritional components of dry beans - - - 15

Table 5: Effect of Processing Method on the Composition of

Bambara Groundnut (BGN) - - - - - - 44

Table 6: Effect of Processing Methods on Some Anti-Nutritional

Factors in Bambara Groundnut - - - - - 45

Table 7: Effect of Processing Methods on Selected Functional

Properties of Bambara Groundnut - - - - - 46

Table 8: Consumer acceptance study of extrudates from treated bambara

groundnut. - - - - - 48

Table 9: Chemical Composition of “Acha” and Carrot (per 100g

sample) - - - - - - - 48

Table 10: Proximate Composition of Un-extruded & Extruded Bambara

- “Acha” Containing Graded Levels of Carrot - - - - 49

Table 11: Mean Sensory Scores of Bambara-“Acha” Extruded Snacks

Containing Graded Levels of Carrots - - -- 51

Table 12: Effect of Extrusion on Anti-Nutrient Content of BGN/Acha

Blends Containing Graded Levels of Carrot - - - 52

Table 13: Effect of Processing on In-vitro Protein Digestibility of

BGN-Based Extruded Snacks Containing Graded Levels of

Carrot - - - - - - 53

Table 14: Effect of Processing on the Mineral Content of Extruded Snacks 56

Table 15: Effect of Processing on ß-carotene and C Contents of the

Extruded Snacks - - - - - - - 56

Table 16: Effect of Extrusion on the Vitamin B Content of Bambara-“Acha”

Extruded Snacks Containing Graded Levels of Carrots (mg/100g) 58

Table 17: Effect of Storage Period on the Texture (Crunchiness)

of Extruded Snacks - - - - - - 58

Table 18: Effect of Storage on the Sensory Qualities of Extruded Snacks

(Stored for 6 Months) - - - - - - - 59

Table 19: Effects of Storage on the provitamin A Content of Extruded Snacks - 61

ix

LIST OF FIGURES

Fig. 1: Flow chart for the production of flour from germinated Bambara

groundnut - 28

Fig. 2. Flow chart for the production of flour from roasted Bambara groundnut - - 29

Fig. 3: Flow chart for the production of extruded snacks from BGN, “acha” and carrot composite flour - - 30 Fig. 4: Effect of storage period on the moisture content of the extruded snacks - 60

x

APPENDICES Page Appendix 1: Phosphorous standard curve -- --- -- -- 74 Appendix 2: Iron standard curve -- -- -- --- -- -- 75 Appendix 3: Magnesium standard curve -- -- --- -- -- 76 Appendix 4: Phytate standard curve -- -- --- -- -- 77 Appendix 5: Vitamin A standard curve -- -- -- -- -- 78 Appendix 6: Vitamin B1 standard curve -- -- --- -- -- 79 Appendix 7: Vitamin B2 standard curve -- -- --- -- -- 80 Appendix 8: Vitamin C standard curve -- -- --- -- -- 81 Appendix 9: Vitamin B6 standard curve -- -- --- -- -- 82

1

CHAPTER ONE

1.0 I N T R O D U C T I O N

In many developing countries such as Nigeria, malnutrition is an endemic dietary

problem characterized by protein-energy malnutrition and micro-nutrient deficiency

(Nnanyelugo, 1990; Bowley, 1995; Adelekan et al., 1997; WHO, 2005, 2006). In the

past few years, efforts have been made to reduce or eliminate the problem globally.

Dietary diversification has been suggested as the ultimate solution to malnutrition

challenges. Dietary diversification involves the use of commonly available or consumed

grains, legumes and other nutritious crops to meet the nutritional/dietary need of the

population. Consequently, there is a need for baseline research to identify and exploit

the potentials of locally available but under-utilized agricultural produce in nutritious

product formulations.

Among the locally available under-utilized agricultural produce are bambara groundnut,

hungry rice (“acha”) and carrot, whose utilization are presently limited to household

level, even though they have potentials for industrial application. Bambara groundnut is

an under-utilized indigenous African legume and one of the most important crops in the

continent. Total production has been estimated to be over 300,000 tons per year

(Poulter, 1981). It is an inexpensive source of high quality protein and the third most

important legume in Africa, after cowpea and groundnut (Obizoba and Egbuna, 1992;

Enwere and Hung, 1996). Despite this, its use is limited to household consumption in

most parts of Nigeria. In Eastern States, the seed is used in the preparation of a steam

gel popularly known as “Okpa” while in the Northern parts it is consumed in the form of

meal or roasted snack. According to Poulter (1981), bambara groundnut contains 24%

protein, 6-8% lysine, 1.3 methionine and 50% carbohydrate, it also contains reasonable

quantities of minerals and vitamins.

Hungry rice commonly referred to as “acha”, “fonio” or “finni” is another under-utilised

crop. It is estimated that over 101.3 tons is produced annually in Nigeria, mostly in the

Northern States (Bauchi, Plateau and Kaduna) (CBN, 2005). Hungry rice is processed

and consumed in a variety of ways such as “tuwo”, “kunnu”, “gote”, while whole grains

are used in preparation of soup and porridge (Jideani, 1999). Hungry rice is reported to

be uniquely rich in methionine and cystine (NRC, 1999). It also relatively evokes low

sugar release on consumption, which is an advantage for diabetics (Ayo et al., 2003).

2

Carrot (Daucus carota) is one of the traditional root crops of Northern Nigeria. It is very

rich in carotene the precursor of vitamin A, and contains appreciable amount of

thiamine and riboflavin (Pederson, 1980). Carrot is fast acquiring the status of “lost

crop” in the African continent because its local utilization is limited to direct eating in

unprocessed form as snack (Pederson, 1980). There is need to diversify and

popularize other means of utilizing carrot to derive maximum health benefit from its

nutrient particularly carotenoids(carotene/β- carotene).

Blends of these nutrient dense agricultural produce could be exploited to develop

nutritious shelf stable snacks, which could help in alleviating problems of protein-energy

malnutrition and micronutrient deficiency prevalent in the country. However, to get

maximum nutrient benefit from these crops, they need to be processed to reduce or

remove inherent anti-nutrients that may interfere with the biological availability of the

nutrients. Among the methods used in removing inherent anti-nutrients include roasting,

germination, frying, cooking and recently extrusion cooking (Siegal and Fawcett, 1976;

Rajawat et al., 1999, Nwabueze, 2006).

Extrusion cooking technology is a high temperature short time (HTST) technology. It

has been extensively used in producing varieties of food products, especially in creation

of novel food products and improvement of existing ones like snacks (Lowtan et al.,

1985; Lasekan et al., 1996). It is considered a beneficial food processing technique,

due to its effective destruction of growth inhibitors and contaminating micro-organisms

(Tarte et al., 1989; Chang et al., 2001). It has also been shown to improve the

nutritional quality of food products like snacks (Pham and Rosario, 1987, Rajawat et al.,

1999; Nwabueze, 2006).

1.1 Statement of the problem

Most developing countries experience high burden of protein – energy

malnutrition and severe micro-nutrient deficiencies, which have attracted the numerous

interventions by major stakeholders, to mitigate the challenges. Dietary diversification

involving the use of commonly available or consumed grains, legumes and other

nutritious crops to meet the nutritional needs of the population has often been

advocated. Consequently, the idea of production of nutrient dense ready-to-eat

extruded snacks from blends of bambara groundnut, hungry rice (“acha”) and carrot

3

appears to be a very attractive strategy to combat the observed nutritional challenges.

However, to get maximum nutrient benefit from these crops, processes like roasting,

germination and then extrusion were employed to reduce/eliminate the inherent anti-

nutrients that may interfere with the biological availability of the nutrients and enhance

acceptability.

1.2 Significance of the study

This research work has the capacity to address the twin problems of protein-energy

malnutrition and micronutrient deficiencies, which pose a challenge to meeting nutrition

related Millennium Development Goals (MGDs). It will stimulate establishment of

facilities for production of nutrient dense ready-to-eat extruded snacks, which could be

readily employed in nutrition intervention program like school feeding programmes,

community nutrition activities, nutrition support in emergency situations and promotion

of food security for vulnerable groups/households.

1.3 Objectives of the study

• To produce high nutrient composite flour from blends of bambara ground nut,

hungry rice and carrot.

• To utilize the composite flour in the production of extruded snacks.

• To evaluate the nutritional quality and sensory properties/acceptability of the

products.

• To study the shelf life of the developed and packaged products.

4

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Extruded Snacks

Snack foods are foods which can be taken in place of or between meals. They are

convenient because they are quick and easy to eat. According to Lasekan et al., (1996)

snack foods add variety to the diet which partially explains their popularity. They may

also play a natural role on special occasions or when offered to visitors. Snack food

consumption has also been on the increase in Nigeria and other parts of the world

(Akpapunam and Darbe, 1994). This is as a result of urbanization and increase in the

number of working mothers. This development is being exploited by researchers and

food industrialists to develop innovative snack products that are nutritious - containing

increased protein, complex carbohydrates, and reduced fat (Shaw et al., 1994). Most

snacks are poor sources of protein, and often of poor nutritional quality. According to

Akpapunam (1984) this is because they are mostly prepared from plant food produce,

especially cereals, and cereal proteins are generally low in lysine and total protein

content, although high in sulphur containing amino acids. According to Bressani et al.

(1962), high protein and improved nutritional quality snacks could be produced by

combining cereals with animal food sources or, better with cheaper and more available

plant protein sources, such as legume and oil seeds.

Extruded snack foods have become an integral part of the eating habits of most of the

world’s population. Extrusion technology, at present, has become one of the major

processes of producing varieties of food or creating new food products and to improve

existing ones (Faubion and Hoseney, 1982; Lawton et al. 1985; Miller, 1993). Megard

et al. (1985), Tarte et al. (1989) and Chung et al. (2001) had applied high temperature

short time (HTST) extrusion processing in the development of snack foods, and

considered it a beneficial food processing technique due to its effective destruction of

growth inhibitors and contaminating microorganism. Texturized vegetable protein

products, which imitate that of muscle tissues have also been produced by extrusion

cooking (Chang et al., 2001). Yuryer et al. (1995) reported the use of isolated soybean

protein (ISP) in extruded snack products. With a model system of ISP and potato

starch, he showed that their blending was marked by improved textural and functional

properties of the extrudates. Camire et al. (1991) investigated the characteristics of

5

extruded mixtures of corn meal and glandless cotton seed flour, and reported that

snacks produced with 12.5% cotton seed flour were as acceptable as those containing

only corn, though extrusion reduced protein solubility and available lysine in the

samples. Bjorck and Asp (1989) observed that extrusion processing conditions have

been found to have a positive impact on nutrient retention. Chigumra (1992)

investigated the use of extrusion cooking technology in the production of maize,

sorghum and soybean based snacks, and reported the development of a nutritious

ready – to – eat high protein snack food products, which showed a marked

improvement on children’s health when consumed in Zimbabwe.

Rajawat et al. (2000) evaluated changes in the functional properties of faba beans flour

upon extrusion cooking at variable extrusion temperatures and feed moisture, and

reported that extrusion process reduced emulsification capacity, fat absorption capacity

and foaming capacity drastically, whilst foam stability was slightly affected. Nwabueze

(2006) studied the effect of hydration and screw speed on the nutritional qualities and

acceptability of extruded ready-to-eat African bread fruit snack, and reported that

acceptable products with adequate nutritional quality and anti-nutritional factors were

produced on extrusion cooking. There is a possibility and potential for making new food

products such as nutritious snacks by extrusion cooking of bambara, which is an

industrially under exploited or under utilised indigenous African legume, either alone or

in combination with other locally grown crops (cereals/tubers).

2.2 LEGUMES

Legume refers to the edible seeds of leguminous plants belonging to the leguminosae

family and they constitute an important source of dietary protein especially for most

people in developing countries (Siegel and Fawcet, 1976). The short fall in the

production of animal proteins and wide prevalence of protein malnutrition in developing

countries of the world have refocused the importance of legumes as source of protein in

human diets, especially in those countries where the consumption of animal protein is

limited by its low availability with its consequent high cost, cultural or religious habits

(FAO,1982). Apart from being an excellent and inexpensive source of dietary protein,

legumes have low sodium and high potassium contents, abundance of complex

carbohydrates, ability to lower serum cholesterol in humans, high fibre and low fat

6

contents, long shelf-life (in dried form) and diversity of the foods that can be made from

them (Sathe, 1996).

The history of grain legume production and consumption, shows that of the legumes

now being cultivated throughout the world, Pea (Psium spp), Cowpea (Vigna

unguiculata), Lentil pea (Cajanus cajan), Bambara groundnut (Voandzia subterranea)

and the Egyptian pea (Tritolium alexandrum) are indigenous to Africa (Idusogie, 1973).

Other legumes of the African continent are groundnut and soybean. The availability

and consumption of legumes in different regions of the world vary greatly. The

production of grain legumes in the world is around 52 MT (Table 1). The African

continent is characterized by moderate to intensive use of legumes. In some African

countries beans consumption is so large that about 65% of calorie intake is in the form

of legumes. Among those legumes, cowpea is grown and consumed most throughout

the continent with 90% of the world production (FAO, 1982). Nigeria accounted for

about 66% of the total world production of cowpea in 1972 (FAO, 1974), and represents

a cheap and important source of dietary protein for Nigerian populace (Apata and

Ologhoho, 1994).

Table 1: Trend in Total Distribution of Major Food Crops in Nigeria (‘000 tons) 1989 – 91 2000 2001 PULSES World 55856 54631 52385 Africa 6731 8000 8392 Nigeria 1363 2200 2200 CEREALS World 1903795 2063521 2086123 Africa 98735 114068 116503 Nigeria 18100 22891 22891 TUBERS World 577470 698169 677926 Africa 112994 164521 165719 Nigeria 35155 66578 66578 CASSAVA World 155264 176784 178868 Africa 72032 94676 95239 Nigeria 20821 33854 33854 Source: FAO (2002)

7

2.2.1 Bambara Groundnut

Bambara groundnut (Vigna subterranea) is an important indigenous Nigerian legume,

other names are: congo goober, earth pea, kaffir pea, ground bean, jungo bean,

Madagascan or stone groundnut or haricot pistache. It is the third most important

leguminous crop in Nigeria after cowpea and groundnuts (FAO, 1982; Doku and

Karikari, 1971; Alobo, 1999). Bambara groundnut (BGN) is one of the earliest grain

legume crops cultivated on the continent of Africa and is probably of West African origin

(Kay, 1979). It thrives in hot sunny climates on sandy loam soils but it is very adaptable

and grows on poor soils and is drought resistant. It is short lived, with 3-5 month

maturity period. It has branched stems and composed of leaves with three leaflet and

about 30 cm high. The pods are hard and wrinkled when dry, with each pod containing

one or two seeds. The seeds may be eaten raw when immature or the immature pods

and seeds may be boiled. The mature seeds are large and have high fat content with a

seed colour varying from black to white, red, cream, brown and may be mottled with

various colours (Oyenuga; 1968; FAO, 1982). The matured seeds are edible when

roasted or boiled or may be ground into flour and used in local dishes. Total production

has been estimated to be over 30,000 tons per year with Nigeria, Upper Volta, Niger,

Ghana, Togo and Ivory Coast, being the major producers (Doku et al., 1978). The

production of BGN in Nigeria has been on the increase. About 400,000 tons of BGN

were produced in Nigeria in 1981 (FAO, 1982). BGN has great potential in addressing

the protein energy malnutrition in Nigeria, if the technology of its industrial utilization is

effectively developed and applied.

2.2.2 Processing of Legumes

Legumes are generally consumed after some kind of processing like steaming,

sprouting, parching, roasting, puffing or along with cereals or tubers (FAO, 1982). Their

preparation techniques generally depend upon the structural and textural needs,

cultural food habits of the people (Siegel and Fawcet, 1976).

2.2.3 Milling and Grinding

Traditionally in Nigeria and other African countries, legumes are milled by mortar and

pestle, hand operated stones and chakkis are used at household level and in the

modern dhal mill respectively (Kurein, 1981). However with the advent of technological

advancement, grinders are commonly used in most localities. Powdered legumes are

8

used in a variety of dishes and savoury preparations. Grinding is done by wet or dry

methods. The relative proportion of particles of different mesh size is a major

influencing factor on the texture and quality of the product (Kurien, 1981).

2.2.4 Soaking, Boiling and Steaming

Soaking is the pretreatment that facilitates husk removal as well as to soften the

legumes and make them easier to cook. Cooking whole legumes in boiling water is the

most common method used in legume food preparation. According to Salunkhe et al.

(1985) soaking results in loss of ash, iron, copper and drastic reduction in flatulence

caused by oligosaccharides. Since excessive cooking repeatedly leads to a lowering of

digestibility, possibly due to the action of amino acid groups with carbohydrates and the

inactivation or destruction of certain essential amino acids, it is important that an

optimum cooking time be used, which will yield an acceptable textured bean product,

possessing the highest nutritive value (Siegel and Fawcett, 1976). Steaming is

primarily used as a secondary process for converting prepared legume flours and paste

into traditional products.

2.2.5 Germination

Germination process involves an initial soaking of the whole legume grain for 24 hours

to enable the grains to absorb enough moisture that would activate the growth

enzymes. This is followed by spreading the soaked grains on a damp cloth for up to 48

hours to provide appropriate condition for the process, and reduce the likelihood of

grains dry out, which will terminate the germination process. Seed constituents present

in an inert form are altered during soaking and germination and become more

assimilable for human nutrition (Kurien, 1981). Although germination doesn’t reduce

cooking time or improve texture, it brings about chemical changes, since it primarily

involves the carbohydrates of the grain, namely the conversion of some starch to lower

molecular disaccharides and dextrins by the action of amylases, which results in a

gradual decrease in the carbohydrate content of pulses during the course of

germination (Siegel and Fawcett, 1976; FAO, 1982). Similarly, protease activity also

increases during germination causing the degradation of high molecular weight protein

to lower ones leading to a noticeable increase in the concentration of amino acids.

Increase in some of the essential amino acids viz. lysine by 24%, threonine by 19%,

9

alanine by 29% and phenyl alanine by 7% has also been reported (Siegel and Fawcett,

1976).

2.2.6 Roasting

Legumes are sometimes roasted, toasted or heated to improve their nutritional value

and taste/acceptability (Kurien, 1981; Salunkhe et al., 1985). Roasted legumes are

generally consumed after mixing with parch or malted cereals or oil seeds. Roasting

brings about changes in aroma which are described as nutty, burnt and coffee -like due

to the formation of pyrazine compounds in the roasted food. It was reported (Powrie

and Nakai, 1981) that the level of pyrazine compounds also related to the extent of

browning. Roasting time is very important in enhancing PER in legumes, about 15

minutes of roasting at 200ºC was the optimum time to maintain maximum protein

quality considering available lysine.

2.2.7 Preparation of Protein Concentrate

Protein concentrates enjoy widespread commercial use in the fortification of foods and

beverages, which increase their nutritional value and improve functionality (Sosulki and

Young, 1979). They are generally prepared by wet extraction methods and their protein

contents normally range between 70-90% and 90-98% by weight. On the other hand,

protein rich flours could also be prepared by air classification, which is a means of

fractionating finely milled legume flour into protein and starch concentrates using a

spiral air stream (Heavier starch granules are separated from the finer protein – rich

particles) (Siegel and Fawcet, 1976). This dry separation technique is more promising

for starchy legumes (Sosulski and Young, 1979). The obtained protein fraction retains

good functional properties and there is no effluent to dispose of. The technique

involves finely milling the seeds by pin-milling and passing the flour into an air-classifier.

This separates (concentrate the protein) the smaller protein bodies from the larger

starchy granules by virtue of their different behaviour in the air flow (based on the

difference in size, shape, and density of the starch granules and the protein –

containing particle). In this way a protein concentrate (fine fraction) and a starch

concentrate (Coarse fraction) are produced. The hull is collected with the coarse

fraction, and the lipid material with the fine fraction (Vose et al., 1976). In a typical pilot

study, pea flour containing 21% fines (pea protein concentrate) with 60% protein

content and a coarse fraction (starch) with 8% protein were obtained. Repeated milling

10

gives an additional 10% pea protein concentrate with lower (45%) protein content

(Siegel and Fawcett, 1976).

2.2.8 Canning of Legumes

Canning represents another common method of preserving legumes especially in

developed countries. The most popular kinds of legumes that are canned are beans

(Phaseolus vulgaris L), navy or kidney beans, and Lima or buffer beans (Phaseolus

lunatus). They are consumed as vegetable side dishes or basic ingredient in salads.

Also, green or garden peas and black eye pea (Vigna unguiculata) are also canned.

Pre-cooked canned beans (Phaseolus vulgaris) are consumed in parts of Latin

America.

2.2.9 Quick Cooking Legumes

Quick cooking legumes are becoming popular since their preparation requires shorter

cooking time. More recent studies in the area of quick cooking dried beans include the

development of an intermittent vacuum treatment (Hydravac process) for 30-60 minutes

in a solution of inorganic salts (sodium chloride, tripolyphosphate, bicarbonate and

carbonate (Rockland and Metzler, 1976). The process consists of loosening the seed

coats by vacuum treatment, hot-water or steam blanching, soaking in solution of in-

organic salts, drying the processed beans under low velocity air stream of below 60ºC

for 24 hours. The overall advantage of this quick cooking process is its conversion of

dry beans to rehydrated product that cooks within 15 minutes, with about 80% reduction

in cooking time of 1-3 hours. Development of an inexpensive mechanical method for

producing quick cooking beans (California small white, Sanilac, Pinto) has been

reported by Kon et. al. (1973). The process of presoaking has been developed for

Pigeon pea by which the cooking time is reduced by 50% (Kon et. Al. 1973).

2.2.10 Nutritional Importance of Legumes

Legumes are important sources of many nutrients (Siegel and Fawcett, 1976; FAO,

1982), such as of protein, vitamins and minerals (Table 2). Their crude protein content

varies and ranges from 18 – 36%, and is of moderate quality, though deficient in

sulphur containing amino acids like methionine and tryptophan (Table 3). The use of

legumes makes a valuable contribution to the protein content of diets based

predominantly on root crops and cereals as staples.

11

TABLE 2: Energy and Selected Nutrients in Some Legu mes

(Composition Per 100g Edible Portion of Dried Matur e Whole Seeds)

Cowpea Bambara Groundnut

Black Gram

Chikpea Groundnut Soy bean

Water % 11.5 10.1 10.6 11.0 7.3 10.2 Protein (g) 22.7 16.0 21.0 19.4 23.4 35.1 Fat (g) 1.6 6.0 1.6 5.6 45.3 17.7 Carbohydrate (g) 61.0 65.0 63.4 60.9 21.6 32.0 Crude fibre (g) 4.2 ND 4.4 2.5 2.1 4.2 Dietary fibre (g) ND ND 19.5 25.6 6.1 11.9 Ash (g) 3.2 3.0 3.4 3.1 2.4 5.0 Energy (Kcals) 340 370 344 362 548 400 Calcium (g) 110 85 110 114 58 226 Iron (mg) 6.2 4.2 8.4 2.2 2.2 8.5 Thiamin (mg) 0.59 0.18 0.58 0.46 1.00 0.66 Riboflavin (mg) 0.22 ND 0.20 0.20 0.13 2.2 Nicotinic acid (mg) 0.22 ND 2.3 1.2 16.8 2.2

Source: FAO (1982)

Table 3: Essential Amino Acid (EAA) Composition of Selected Legumes (g/16g)

EAA Cowpea Bambara ground-

nut

Black gram

Chikpea Ground -nut

Soybean FAO scoring pattern

Isoleucine 3.8 4.4 4.3 4.4 3.5 4.5 4.0

Leucine 7.0 7.8 7.8 7.5 6.4 7.8 7.0

Lysine 6.8 6.4 7.4 6.8 3.5 6.4 5.0

Methionine 1.2 1.8 1.4 1.0 1.2 1.3

3.5 Cysteine 1.1 1.0 1.0 1.2 1.2 1.3

Phenyl alanine 5.2 5.6 6.6 5.7 5.0 4.9 6.0 Tyrosine 2.6 3.5 3.4 2.9 3.9 3.1

Threonine 3.6 3.3 3.7 3.8 2.6 3.9

4.0

Tryptophan 1.1 1.1 ND ND 1.0 1.3

1.0

Valine 4.5 5.3 5.9 4.5 4.2 4.8

5.0

Source: FAO (1982); ND = Not determined

Most of the legumes contain about 2% fat on the average though bambara groundnut

has up 5-7% fat. Soya beans, groundnut and some other legumes have up to 18-48%,

and are regarded as oil seeds. These legumes are also good sources of minerals such

12

as calcium, iron, copper, zinc, potassium and magnesium, which contributes 25-30% of

the total mineral content of legumes, and can be beneficially utilized in the diets of

people who take diuretics to control hypertension and who suffer from excessive

excretion of potassium through body fluids (Salunkhe et al., 1985). They also provide

variable quantities of most vitamins depending on the stage of maturity and state of

dryness. Beta-carotene, thiamine, riboflavin, niacin and ascorbic acid are present in

many legumes in appreciable amounts.

2.2.11 Antinutritional Constituents of Legumes

Although legumes are good source of protein, carbohydrates and minerals, they remain

under-exploited as human food due to the presence of several antinutritional

constituents, which lower the protein quality as compared to animal proteins. Some of

these anti-nutritional constituents and their deleterious effects are given in Table 4. The

major antinutritional constituents found in legumes are protease inhibitors, phytates,

lectins, amylase inhibitors, polyphenols, cyanogens and others. A brief description of

these are given below

2.2.11.1 Protease (trypsin) inhibitor

Enzyme inhibitors that can occur naturally in the human body include the pancreatic,

proteolytic inhibitors, blood clotting enzyme inhibitors, liver nicotinamide deamidase

inhibitors, and hyaluonidase inhibitors. Trypsin inhibitors are the most widely distributed

among the protease inhibitors. In addition to trypsin inhibitor chymotrypsin, subtilin,

elastase, plasmin, kallikrein and papain inhibitors are also present (Whitaker et al.,

1973). Two main trypsin inhibitors Bowman Birk and Kunitz are isolated from legumes.

These inhibitors lower the nutritional value of protein. Rackis (1974) and Liener and

Kakade (1980) have reviewed their nutritional implications in diets.

The trypsin inhibitors are present in some of the common foods like soyabean,

limabean, Phaseolus aureus, cowpea, navy bean, kidney and blackbean, broadbean

(Vicia faba), double bean, field beans, (Dolichos lablab), bambara groundnut, jackbean

(Canavalia ensiformis), pigeon pea (Cajanus cajan), other foods like cereals (wheat

flour), tubers, vegetables, nuts and eggs (Mathews, 1989). The trypsin inhibitors

adversely affect the digestion of dietary protein by proteolytic enzymes present in the

intestinal tract. The growth depression caused by the trypsin inhibitors may be the

13

consequence of an endogenous loss of essential amino acids being secreted by a

hyperactive pancreas, since pancreatic enzymes such as trypsin and chymotrypsin

particularly rich in sulfur containing amino acids resulted in pancreatic hypertrophy and

amino acid requirements (Salunkhe et al., 1985). Various processing methods such as

toasting, cooking, germination, fermentation, germination and extrusion decrease the

trypsin inhibitor activity (Bodwell and Hopkins, 1985; Kakade et. al., 1974, Apata and

Ologhoho, 1997; Wang et. al. 1997; Nwabueze, 2006; Singh, 2007).

2.2.11.2 Phytates

Phytic acid, the hexaphosphate of myoinositol is found in various legumes and also in

other foods which contain larger amounts of phytates. The phytate in seeds is

concentrated primarily in the bran and germ. Phytate has been generally regarded as

the primary storage form of both phosphorous and inositol in almost all seeds. In food

such as legumes and grains, a large proportion (60-80%) of phosphorous is present in

the form of phytic acid primarily as a complex salt of divalent minerals such as zinc,

calcium, magnesium and iron or as complexed with proteins. It has been shown that

phytate is involved in lowering the bioavailability of minerals (Salunkhe et al., 1985;

Oberleas, 1973). Phytate also interacts with protein resulting in reduced protein

solubility which affects their functional properties. It has been shown that phytate

inhibits several enzymes including pepsin, α-amylase and trypsin. Various processing

methods such as malting, germination and blanching reduce the phytic acid by 38-46%.

Reduction of phytic acid during blanching and malting may be due to leaching of

phytate ions into the soaking medium and increased activity of phytase enzyme during

germination (Archana and Kawata, 1998; Salunkhe et al. 1985).

2.2.11.3 Polyphenols (Tannins):

Polyphenols have been recognized as one of the antinutritional factors in legumes.

They are mainly located in seed coat with low or negligible amount in cotyledons.

Polyphenols are known to interact with proteins leading to either inactivation of

enzymes such as trypsin or chymotrypsin making the proteins insoluble. Polyphenolic

compounds inhibit other hydrolytic enzymes such as α-amylase and lipase, and are

known to decrease the digestibility of carbohydrates, protein and bio-availability of

vitamins and minerals (Salunkhe et. al. 1985). The major phenolic compounds of

concern are flavonoids, coumarins and furomarins, tannins and gossypols (Doyle et. al.

14

1994). Tea polyphenols, which were previously found to inhibit human salivary

amylase, have also been shown to inhibit sucrase and α-glucosidase. Dietary tannins

significantly decreased the digestion of protein and increased feed nitrogen extraction

but did not affect the biological value of nitrogen in the diet (Doyle et. al. 1994).

2.2.11.5 Cyanogens

These are substances capable of producing cyanide in plant foods. Cyanide

compounds are widespread throughout plant kingdom. They occur mainly in the form

of glycoside. They can be broken down to cyanide and the sugar residue by the action

of the enzyme glucosidase present in the plant (Montgomery, 1980). Lima bean has

the highest concentration of cyanogens (Linamarin) of all the food legumes (FAO,

1982), and cassava has the highest level of cyanogens among various fiber crops and

this is distributed throughout the plant (Balagopalan et. al., 1988). Large doses of

cyanide cause death by inhibition of cell respiration, but in small doses are converted

on ingestion into thiocyanate, a well known goitrogen. Although most food legumes

contain only low levels of goitrogens, the conversion of cyanogens into goitrogens may

explain some of the aetiology of goiter in certain areas of the world (FAO, 1982;

Mathews, 1989).

Legume seeds contain flatulence-producing oligosaccharides such as raffinose,

starchyose and verbascose. These sugars are not digested in the small intestine as

appropriate enzymes are lacking, so they pass to the large intestine where bacteria

fermentation result in gas production (Salunkhe et al., 1985; Augustine and Klien, 1989)

causing inconvenience. Rackis (1974) showed that the bacteria responsible for the

production of gas were spore-forming clostridia present in the colon. Flatulence is one

of the important factors limiting the consumption of bambara groundnut and other

legumes.

2.2.11.6 Lectins

Lectins are proteins that have the ability to clump or agglutinate and break down the red

blood cells in a way similar to antibodies. Lectins apart from causing food poisoning,

lower the protein quality of foods and cause reduction in rate of growth when they are

fed to experimental animals (FAO, 1982). However, the phyto haemagglutinins are

heat labile. Normal cooking destroys their specific action, cooking of beans for one

15

hour at 100oC destroys the toxicity and haemagglutinating activity completely (Jaffe,

1973).

Table 4: Effects of Antinutritional components of d ry beans

Components Effects

• Trypsin inhibitors Trypsin inhibition, pancreatic hyperotrophy, dietrary loss of lysine

• Chymotrypsin inhibitors Chymotrypsin inhibition

• α-amlyase inhibitors α-amylase inhibition, hinders carbohydrate utilization

• Subtilin inhibitors Subtilin inhibition

• Phyto-haemaglutinins (Lectins) Growth depression, fatal

• Phytates Reduced metal bioavailability, altered protein stability

• Flatulence factors Flatulence (Hydrogen, carbon dioxide and methane) production.

• Polyphenols Reduction in protein digestibility, inhibition of several enzymes.

• Off flavours Damage to amino acids, renders unacceptable to consumers

• Phytoalexine Hemolysis, uncouple oxidative phosphorylation

• Cyanogens Hemolysis, uncouple oxidative phosphorylation

• Goitrogens Inhibition of iodine binding to thyroid gland

• Lathyrism Nervous paralysis of lower limbs, fatal

• Favism Hemolytic anemia

• Allergens Several allergenic reactions bitter taste, foaming, hemolysis

• Saponins Growth inhibition, interference with reproduction performance.

• Estrogens Rachitogenic

Antivitamins of • Vitamin D • Viatamin E • Vitamin B-12 • Lysinoalanine

Liver necrosis, oxidation of vitamin E, muscular Dystroyphy Increased vitamin B-12 requirements Nephrotoxicity, reduction in available lysine, kidney cell, nucleus and cytoplasm enlargement. Generation of D-amion acids, act as synergist to lysinoalanine in the expression of nephrocytomegaly.

Source: Salunkhe et al. (1985)

2.2. 11. 7 Effect of processing on legume food qua lity

Processing conditions bring about changes in the nutritional quality of legume proteins,

their functional properties and anti-nutritional factors. The effect may be beneficial or

adverse depending on the conditions used. Some of the beneficial effects include

imparting attractive aroma, flavour, improvement of digestibility and palatability and

16

some functional properties and reducing/eliminating some of the antinutritional

components. On the other hand, processing may also have some deleterious effect on

legume proteins. The protein quality is primarily dependent on its essential amino acid

composition. Processing may result in the actual destruction of amino acids or binding

of amino acids due to the formation of linkages not hydrolyses by digestive enzymes

(Bender, 1972) or racemization of amino acids. Also processing methods which expose

the proteins to different conditions can cause changes in their structure/conformation

and may result in a loss or alteration of functional properties. The functional properties

are influenced by a number of factors such as the source of protein, method of isolation

and precipitation drying/dehydration, heating, concentration, chemical or enzymatic

modifications, environmental factors like temperature, pH and ironic strength.

Dry legume seeds normally require relatively long time to cook. Seeds of broadbean,

chickpea, bambara groundnut, common bean and to a lesser extent lentils are soaked

in water overnight before cooking as a means of reducing the cooking time. In some

instances small amounts of NaHCO3 is added while cooking to reduce it further. The

cooking process soften the hard legume seeds, improve the plasticity of the cell walls

thereby facilitate cell expansion and reduction of cellular adhesion. Some legume

seeds are very difficult to cook due to the presence of insoluble pectins in the form of

calcium and magnesium pectate. It has been reported that cooking quality may be

associated with the ratio of monovalent to divalent cations and with the phytic,

phosphorous, lignin and α-cellulose content of the seed. Cooking quality has been

found to be affected by storage conditions and moisture content of the seed.

Certain classes of legumes improve their protein value as a result of heat processing,

either through inactivation of trypsin inhibitors or increase the availability of certain

amino acids, particularly the sulphur containing ones. Processing at low temperature

treatments of protein may increase the digestibility (FAO, 1982). The processing

temperature and duration of heating and moisture content are the principal factors in

determining the degree of alteration. Dry heating is less effective than steaming

enhancing nutritive value (FAO, 1982). Negi et al. (2001) reported improved /increased

protein and starch digestibility in moth beans with soaking, dehulling, germination and

pressure cooking. Improved starch digestibility in fermented legumes (Bengal gram,

Cowpea and Green gram) due to breakdown of starch to sugars and reduction in

17

phytate levels has been reported (Urooj and Puttraj, 1994). However, when legumes

are roasted, a decrease in starch digestibility is noticed, perhaps, due to the presence

of enzyme inhibitors. Digestibility is affected by different methods of processing and

cooking. Products that are refined or isolated proteins and well processed have been

shown to have improved digestibility (Bodwell and Hopkins, 1985). El-Adawy et al.

(2000) reported increase in protein solubility, in-vitro protein digestibility and available

lysine and decrease in trypsin inhibitor, phytic acid, tannin and haemaglutinin activity

and carbohydrate with soaking of soybean, lupin seed, bean and black gram

respectively. Umoren et al. (1997) reported improvement in protein quality, reduction in

phytic, tannic acids and elimination of trypsin inhibitor, haemagglutinin and HCN in

cowpeas autoclaved for 30 mins at 105oC and 15 psi. Also germination of cowpeas

improved the in-vitro protein quality and starch digestibility but has little effect on the

overall amino acid profile (Jirapa et al., 2001)

2.2.11.8 Effect of processing methods on antinutr ients

Several methods of processing beans, which include dehulling, milling, soaking,

cooking, germination, fermentation, autoclaving, roasting, frying and parching, protein

extraction and extrusion will reduce/eliminate the antinutrients depending on the type of

bean. Generally adequate heat processing inactivates the protease inhibitors (FAO,

1982). Water soluble compounds such as the raffinose (oligosaccharides) can be

removed to a significant extent by discarding the soaked water, which helps to reduce

the flatulence potential of beans that contain them like bambara groundnut and cowpea

(Sathe, 1996; Smart, 1976). Heat stable compounds such as polyphenols and phytates

are however not easily removed by simple soaking. In such cases, other methods that

can hydrolyse these compounds may be more useful. According to Sathe (1996) after

4 days of germination of chickpea 91% of verbascose and stachyose and 88% of

raffinose were removed.

Soaking and dehulling reduced the phytic content up to 4% in faba bean. Soaking and

cooking, soaking, dehulling and cooking reduced the phytic acid content by 32-35% in

different varieties of faba bean. Autoclaving and germination reduced up to 55 and

69%, respectively (Sharma and Seighal, 1992).

The processing methods such as steaming and fermentation had been shown to

reduce the trypsin inhibitor activity of soybean (Doyle et al., 1994). Autoclaving of

18

cowpea for 30 minutes at 105oC and 15 psi resulted in total inactivation of the trypsin

inhibitor, haemagglutinin and HCN activities (Umoren et al. 1997). Total inactivation of

trypsin inhibitor had also been reported with autoclaving of black gram at 20 lbs for 20

minutes (Hajela et al, 1998). However, roasting showed only 6% destruction while

about 10% chymotypsin inhibitor activity remained. Inactivation of trypsin inhibitor is

greatly influenced by moisture content of food, time and temperature and methods of

heating and drying (Bodwell and Hopkins, 1985; Hajela et al. 1998).

Malting for 72 hours was found to be the most effective method in decreasing (40%) the

polyphenol content. Loss of polyphenol during malting was attributed to the presence

of polyphenol oxidase and to the hydrolysis of tannin protein and tannin – enzyme

complexes, which resulted in the removal of tannins. Germination has also been

reported to reduce the polyphenol content in Pearl millet (Doyle et al. 1994; Archana

and Kawata, 1998).

2.3.0 EXTRUSION COOKING AND ITS EFFECT ON FOOD QUAL ITY

Health and nutrition are the most demanding and challenging fields in this era and

would continue to be in the future as well. Maintaining and increasing nutritional quality

of food during food processing are always potentially important area for research.

Deterioration of nutritional quality, owing to high temperature, is a challenging problem

in most traditional cooking methods. Extrusion cooking is preferable to other food –

processing techniques in terms of continuous process with high productivity and

significant nutrient retention, owing to the high temperature and short time required

(Guy, 2001). Extrusion cooking is a high temperature, short-time process in which

moistened, expansive, starchy and/or proteinacious food materials are plasticised and

cooked in a tube by a combination of moisture, pressure, temperature and mechanical

shear, resulting in molecular transformation and chemical reactions (Havck and Huber,

1989; Castells et al. 2005). This technology has some unique positive features

compared with other heat processes, because the material is subjected to intense

mechanical shear. It is able to break the covalent bonds in biopolymers, and intense

structural disruption and mixing to facilitate the modification of functional properties of

food ingredients and/or texturizing them (Asp and Bjorck, 1989; Carvallo and Mitchelle,

2000).

19

In addition, the extrusion process denatures undesirable enzymes; inactivates some

antinutritional factors (trypsin inhibitors, haemagglutinins, tannins and phytates);

sterilizes the finished product; and retains natural colours and flavours of foods

(Fellows, 2000; Bhandari et al., 2001).

2.3.1 Application of Extrusion Cooking

The process had found numerous applications, including increasing numbers of ready-

to-eat cereals; salty and sweet snacks; co-extruded snacks; indirect expanded

products; croutons for soups and salad; an expanding array of dry pet foods and fish

foods; textured meat-like materials from defatted high-protein flours; nutritious

precooked food mixtures for infant feeding; and confectionery products (Harper, 1989;

Eastman et al., 2001).

2.3.2 Advantages of Extrusion Cooking

Parallel to the increased applications, interest has grown in the physico-chemical,

functional and nutritionally relevant effects of extrusion processing. Prevention or

reduction of nutrient destruction, together with improvements in starch or protein

digestibility, is clearly of importance in most extrusion applications. Nutritional concern

about extrusion cooking is reached at its highest level when extrusion is used

specifically to produce nutritionally balanced or enriched foods, like weaning foods,

dietetic foods, and meat replacers (Cheftel, 1986; Plahar et al., 2003). Many

researchers had reported the positive and negative effects of extrusion process on the

nutritional quality of food and feed mixtures using different extruder conditions

(temperature, feed moisture, screw speed and screw configuration) and raw material

characteristics (composition, particle size).

2.3.3 Effect of Extrusion Cooking on Protein

Protein nutritional value is dependent on the quality digestibility and availability of

essential amino acids. Digestibility is considered as the most important determinant of

protein quality in adults, according to FAO/WHO/UNU (1985). Protein digestibility value

of extrudates is higher than non-extruded products; the possible cause might be

denaturation of proteins and inactivation of antinutritional factors that impair digestion.

The nutritional value in vegetable protein is usually enhanced by mild extrusion cooking

conditions, owing to an increase in digestibility (Hakansson et al., 1987; Colonna et al.,

1989; Areas, 1992). It is probably as a result of protein denaturation and inactivation of

20

enzyme inhibitors present in raw plant foods, which might expose new sites for enzyme

attack (Colonna et al., 1989). All processing variables have different effects in protein

digestibility.

2.3.4 Effect of Extrusion Cooking on the Antinutr ients

An advantage of extrusion cooking is the destruction of antinutritional factors, especially

trypsin inhibitors, haemagglutins, tannins and phytates, all of which inhibit protein

digestibility (Bookwalter et al., 1971; Armour et al., 1998; Alonso et al., 1998, 2000a).

The destruction of trypsin inhibitors increases with extrusion temperature and moisture

content. At constant temperature, inactivation increases with increasing product

residence time and moisture content. The highest protein quality (as measured by

protein efficiency ratio), corrected for a value for casein of 2.5 is 2.15 in extruded soy

flour, obtained at a barrel temperature of 153°C, 20% moisture and 2 min residence

time, coinciding with 89% reduction of trypsin inhibitors(Bjorck and Asp, 1989).

Extrusion (300-r.p.m. screw speed, 27kg/hour feed rate, 5/32 inches die size and 93-

97°C outlet temperature) causes complete destruction of trypsin inhibitor activity in

extruded blends of broken rice and wheat bran containing up to 20% wheat bran (Singh

et al., 2000). However, in blends containing bran beyond 20%, the inactivation of

trypsin inhibitor decreases from 92 to 60% (Singh et al., 2000). This may be correlated

to a lower degree of expansion of extrudates with an increased proportion of bran in the

blends, which probably reduced the effect of heat, resulting in a lower degree of

inactivation of trypsin inhibitor. Lectin (haemagglutinating) activity is relatively heat

resistant. An aqueous heat treatment, at 60 or 70°C for up to 90 min, does not alter the

lectin activity in soybean. Lectin activity is reduced, but not abolished by heating at 80

or 90°C. However, as found with kidney bean (Grant et al., 1982, 1994), the lectin

activity in the fully imbibed seed could be completely abolished by heating them for 5

min at 100°C. Extrusion has been shown to be very effective in reducing or eliminating

lectin activity in legume flour (Alonso et al., 2000b). Thus, extrusion cooking is more

effective in reducing or eliminating lectin activity compared with traditional aqueous heat

treatment. The enzyme hydrolysis of protein is improved after extrusion cooking as a

result of the inactivation of antitrypsin activity in extruded snacks. The higher

susceptibility of protein to pepsin, as compared with trypsin, further suggested the

presence of antitrypsin activity. The improvement in pepsin hydrolysis might be the

result of the denaturation of proteins during extrusion cooking, rendering the more

21

susceptible to pepsin activity. This suggests that extrusion considerably improved the

nutritive value of proteins (Singh et al., 2000).

2.3.5 Effect of Extrusion Cooking on Amino Acids

Among all the essential amino acids, lysine is the most limiting essential amino acid in

cereal-based products, which are the majority of extruded products. Thus a focus on

lysine retention during the extrusion process is of particular importance.

The available lysine in the extrudates of defatted soy flour and sweet potato flour

mixture ranged from 68 to 100% (Iwe et al., 2004). Increase in screw speed (80-

140r.p.m) and a reduction of die diameter (10-6mm) enhance lysine retention. Even

though an increase in screw speed increases shear, leading to more severe conditions,

the corresponding reduction in residence time (as a result of increase in screw speed)

limits the duration of heat treatment, resulting in high lysine retention. An increase in

level of sweet potato increases lysine retention, which can be attributed to the lower

levels of lysine in the sweet potato raw material, as the losses are more pronounced at

increasing levels of soy addition, which apparently has higher lysine content. Optimum

available lysine was estimated at a feed composition of 98.49%, screw speed 118.98

r.p.m, mixtures of defatted soy flour and sweet potato flour (Iwe et al., 2004). In the

extrusion of wheat flour (150°C mass temperature, 5mm die diameter, 150-r.p.m screw

speed), an increase in feed rate (from 200-350g min-1) significantly improved lysine

retention (Bjorck and Asp, 1989).

A number of studies suggests that higher moisture content (15-25%) significantly

improved lysine retention (Noguchi et al., 1982; Bjorck and Asp, 1989). It was found

that, at a given process temperature during extrusion cooking of cowpea and mung

bean, the available lysine decreased with increasing feed moisture content at 93 -

167°C barrel temperature, 30-45% feed moisture and 100 to 200-r.p.m. screw speed

(Pham and Del Rosario, 1984). Owing to the complex nature of interactions between

extruder conditions, these changes might not be related to a single factor.

Apart from lysine, a few other amino acids have been affected by a decrease in

moisture content during extrusion. Cysteine decreases below 14.5% moisture content

during the extrusion (181-187°C mass temperature, 12-25% feed moisture, 35 to 79 –

Nm torque) of maize grits (Iwe et al., 2001). Biological evaluation also revealed a

22

decrease in the availability of aspartic acid, tyrosine and arginine with decreasing

moisture content. With increasing energy input to the extruder, a significant reduction in

the availability of several amino acids was found. The loss of available arginine (21%),

histidine (15%), aspartic acid (14%) and serine (13%) was significant at 135-160°C

mass temperature and 150 or 200-r.p.m. screw speed (Iwe et al., 2001). Extrusion

cooking of a cereal blend resulted in a considerable loss of arginine, and to a lesser

extent also of histidine (170°C mass temperature, 10% feed moisture and 40-r.p.m

screw speed). Lysine and methionine availability was not affected below 149°C during

extrusion cooking of soybeans (127-154°C mass temperature, 12% feed moisture and

20-s residence time). At the highest temperature, lysine showed the greatest loss

(31%), although a 13% decrease in methionine was noted (Bjorck and Asp, 1989).

2.3.6 Effect of Extrusion Cooking on Maillard Rea ction

Maillard reaction is a chemical reaction involving amino groups and carbonyl groups,

which are common in foodstuffs, and leads to browning and flavour production. The

nutritional significance of Maillard reaction is most important for animal feeds and foods

intended as the sole item in a diet (Fukui et al., 1993). Maillard reaction occurs between

free amino acid groups of protein and carbonyl groups of reducing sugars, and lead to a

decrease in the availability of amino acids involved and in protein digestibility. Pentoses

are most reactive, followed by hexoses and disaccharides. For hexoses, the order of

reactivity is D-galactose> D-mannose> D-glucose. Reducing disaccharides are

considerably less reactive than their corresponding monomers. Basic amino acids are

more reactive than natural or acid amino acids (Kroh and Westphal, 1989). Lysine

appears to be the most reactive amino acid, owing to the fact that it has two available

amino groups (O’Brien and Morrissey, 1989). Furthermore, lysine is limiting in cereals,

and loss in availability would immediately result in a decrease in protein nutritional

value. Lysine may thus serve as an indicator of protein damage during processing.

However, arginine, tryptophan, cysteine and histidine might also be affected (Iwe et al.,

2001).

The process conditions used in extrusion cooking – high barrel temperatures and low

feed moistures are known to favour the Maillard reaction. In the extrusion cooking of a

cereal mixture, the loss of available lysine range from 32% to 80% at 70°C mass

temperature, 10-14% feed moisture and 60-r.p.m. screw speed (Beaufraud et al.,

23

1978). There was a substantial loss (32%) of available lysine without addition of sugars

in the cereal mixture, which might be the result of hydrolysis of starch. Free sugars

might be produced from starch hydrolysis during extrusion to react with lysine and other

amino acids with free terminal amines. It was found that retention of available lysine

during processing of a cereal/soy-based mixture containing 20% sucrose ranged from

0% to 40% at 170°C mass temperature, 10-14% feed moisture and 60-r.p.m. screw

speed (Noguchi et al., 1982). The loss depends on extrusion conditions, increasing with

temperature and decreasing with moisture content of the feed. In order to keep lysine

losses within an acceptable range, it is necessary to avoid extrusion cooking above

180°C at water contents below 15%, and/or avoid the presence of high amount of

reducing sugars during the extrusion process. Apart from lysine, limited data are

available about the effects of the Maillard reaction on other essential amino acids

during the extrusion process. It is known that the loss of amino acids, owing to the

Maillard reaction is affected by the degree of reactivity of different sugars.

2.3.7 Effects of Extrusion Cooking on Carbohydrates

Carbohydrates range from simple sugars to more complex molecules, like starch and

fibre. Sugars, such as fructose, sucrose and lactose are great sources of quick energy.

They provide sweetness and are involved in numerous chemical reactions during

extrusion. Control of sugars during extrusion is critical for nutritional and sensory quality

of the products. Extrusion conditions and feed materials must be selected carefully to

produce desired results. For example, a weaning food should be highly digestible, yet a

snack for obese adults should contain little digestible material (Camire, 2001).

Several researchers (Noguchi et al., 1982; Camire et al., 1990; Borejszo and Khan,

1992) have reported sugar losses in extrusion. In the preparation of protein enriched

biscuit, 2-20% of the sucrose was lost during extrusion at 170-210 °C mass

temperature and 13% feed moisture (Noguchi et al., 1982; Camire et al., 1990). It may

be explained based on the conversion of sucrose into glucose and fructose (reducing

sugars), and loss of these reducing sugars during Maillard reaction with proteins.

Oligosaccharides (raffinose and stachyose) can induce flatulence and therefore impair

the nutritional utilization of green legumes (Omueti and Morton, 1996). Raffinose and

stachyose contents decreased significantly in extruded high starch fractions of pinto

beans (Borejszo and Khan, 1992).

24

2.3.8 Effect of Extrusion Cooking on Vitamins

The daily vitamin intake might be small compared with other nutrients but the small

quantities consumed are crucial to good health because of the role of vitamins as co-

enzymes in metabolism. The increase in the consumption of extruded infant foods and

similar products which may form the basis of an individual’s diet has focused concern

on the effect of extrusion on the retention of vitamins and minerals that are added prior

to extrusion. As vitamins differ greatly in chemical structure and composition their

stability during extrusion is also variable. The extent of degradation depends on various

parameters during food processing and storage e.g. moisture, temperature, light,

oxygen, time and pH. Among the lipid soluble vitamins, vitamin D and K are fairly

soluble. Vitamin A and E and related compounds- carotenoid and tocopherols

respectively, are not stable in the presence of oxygen and heat (Killeit, 1994). Thermal

degradation appears to be the major factor contributing to β-carotene losses during

extrusion. Higher barrel temperatures (200°C compared with 125°C) reduce all trans-β-

carotene in wheat flour over 50% (Guzman-Tello and Cheftel, 1990).

Ascorbic acid (vitamin C) is also sensitive to heat and oxidation. This vitamin decreased

in wheat flour when extruded at a higher barrel temperature at fairly low (10%) moisture

(Anderson and Hedlund, 1990). Blue-berry concentrate appeared to protect 1% added

vitamin C in an extruded breakfast cereal compared with a product containing just corn,

sucrose and ascorbic acid (Chaovanalikit, 1999). When ascorbic acid was added to

cassava starch to increase starch conversion, retention of over 50% occurred at levels

of 0.4-1.0% addition (Sriburi and Hill, 2000).

In summary, the retention of vitamins in extrusion cooking decreases with increasing

temperature, screw speed and specific energy input. It also decreases with decreasing

moisture, feed rate and die diameter. Depending on the type of vitamin, considerable

degradation can occur, especially in products with high sensory appeal. The following

options for the nutritional enrichment of extruded products with vitamins are possible:

1. The usage of specific vitamin compounds or forms of application with improved

stability;

2. Addition of extra amount to compensate for losses during extrusion and storage;

3. Post extrusion application, e.g. by dusting, enrobing, spraying, coating or filling

together with other ingredients.

25

2.3.9 Effect of Extrusion Cooking on Minerals

Extrusion cooking generally affects macromolecules. Smaller molecules may be

impacted upon by either the extrusion process itself or by changes in larger molecules,

which in turn affect other compounds present in the food. Despite the importance of

minerals for health, relatively few studies have examined mineral stability during

extrusion because they are stable in other food processes (Camire et al., 1990).

Minerals are heat stable and unlikely to become lost in the steam distillate at the die.

Extrusion can improve the absorption of minerals by reducing other factors that inhibit

absorption. Phytate may form insoluble complexes with minerals and eventually affect

mineral absorption adversely (Alonso et al., 2001). Extrusion hydrolyses phytate to

release phosphate molecules. Extrusion of peas and kidney beans resulted in phytate

hydrolysis, which explains the higher availability of minerals after processing (high

temperature extrusion) (Alonso et al., 2001). Extrusion does not significantly affect

mineral composition of pea and kidney bean seed, except for iron. Iron content of the

flours is increased after processing and it is most likely due to the leaching of metallic

pieces, mainly screws, of the extruder (Alonso et al., 2001).

26

CHAPTER THREE

MATERIALS AND METHODS 3.1 Materials

The seeds of Bambara Groundnut (BGN) were purchased from Nsukka market together

with other needed raw materials like “acha”, carrots, sugar, fat, salt and spices. High

density polyethylene for packaging the products was purchased from Polyproducts Ltd,

Ilupeju, Lagos.

3.2. Methods

3.2.1 Preparation of Samples

Bambara groundnut seeds (12kg) were cleaned by sorting and winnowing prior to

sharing into four lots designated A, B, C, and D and were either germinated, roasted,

germinated and roasted, and unprocessed, respectively as described below.

Germination (A) : Germination of BGN seeds was carried out by soaking 4kg BGN

seeds which has been washed with water and soaked for 12hrs at an average room

temperature of 28±20C. After soaking, the grains were spread on wet jute bags and

covered with moistened muslin cloth to germinate. Germinated seeds were removed

after 48hrs, and dried in an air oven (Gallenkamp) at 600C for about 12hrs. The

vegetative parts of the dried BGN were removed by rubbing between palms and

winnowed. The cleaned BGN were milled using Apex mill to pass through 0.4mm mesh

size.

Roasting (B) : The BGN seeds were roasted at 140°C for 40min in an oven (Memmert

GmBH model KG 8540), cooled and milled using Apex mill to pass through 0.4mm

mesh size.

Germination and Roasting (C): Germination and roasting of the BGN seeds were

carried out as described above.

Unprocessed Raw BGN (D): The lot D sample of the BGN was left unprocessed, to

serve as control.

27

Extrusion: Sample (3.6kg) from each lot was mixed with fat (0.36kg), sugar (0.6kg),

salt (0.12kg), spices (0.12kg), and extruded using a single screw extruder (Brabender

PL2001, Germany) at 150°C with screw speed of 170rpm and feed moisture of 20%.

Consumer Acceptance Study

Consumer preference test was done - to select the best treatment for composite flour

production and fortification - by a taste Panel of 50 people to ensure a more accurate

representation of the most preferred treatment. They rated the products attribute of

colour, taste, flavour and overall acceptability on a 9-point hedonic scale, where “9”

represents extremely acceptable while “1” represents extremely unacceptable score.

From preliminary consumer preference test, the roasted sample was then adopted in

producing composite of bambara and “acha” flour, which was mixed with graded levels

of carrot.

Preparation of “Acha” Flour : “Acha” grains (3kg) were sorted, thoroughly washed and

strained to remove sand and other extraneous materials, dried in a Gallenkamp oven at

60°C for 6hrs, cooled and milled using Apex mill to pass through 0.4mm mesh size.

Preparation of Carrot Powder : Cleaned carrots (6kg) were manually grated, dried

(55°C for 8hrs), cooled and milled using Apex mill to pass through 0.4mm mesh size.

Extruded Snacks Formulation and Extrusion : Roasted BGN milled into flour (3.6kg)

and acha flour (1.2kg) composite, were fortified with carrot powder at 0%, 5%, 10%,

15% and 20% level on replacement basis. The samples were mixed with fat (0.36kg),

sugar (0.6kg), salt (0.12kg) and spices (0.12kg), and extruded under similar condition

as previously described. The extruded samples were cooled and packaged in high

density polyethylene sachets for analyses and storage study.

Texture Analysis : The objective texture analysis of the extruded snacks was

conducted with the aid of a Universal Testing Machine (Testometric AX, M500-25KN

England). Each sample was placed on the compression plate and the lever carrying a

suitable mouthpiece was lowered till the sample was crushed. The maximum

compression force attained by each sample, which was displayed on the panel was

recorded.

28

Sensory Evaluation

Organoleptic properties of the samples were evaluated by 20 semi trained panelist for

various sensory attributes using a 9-point hedonic scale questionnaire (Larmond,

1977), where “9” represents extremely acceptable while “1” represents extremely

unacceptable score. The attributes evaluated were crispiness/crunchiness, taste,

flavour, texture, colour and overall acceptability. Data were analysed statistically using

analysis of variance (ANOVA) and mean separation was done by Duncan (1955)

multiple range tests at 5% level of probability.

Storage Study: Samples were stored for six months under ambient conditions

(28±2ºC) and analysed at 2 months interval for moisture, vitamin A and sensory

properties (as described above). The texture of stored samples was also determined

using the Universal Testing Machine (Testometric AX, M500-25KN).

Bambara groundnut (BGN) Seeds

Sorting/Washing

Soaking

Germination (48hrs)

Drying

Derooting/Dehulling

Milling

Sieving

Flour

Fig. 1: Flow chart for the production of flour from germinated BGN

29

Bambara groundnut (BGN) Seeds

Sorting/Washing

Weighing

Roasting (140°C for 40min)

Cooling

Milling

Sieving

Flour

Fig. 2: Flow chart for the production of flour from roasted BGN

30

Composite flour

(Bambara groundnut (BGN), “acha” and carrot)

Weighing

Mixing of ingredients (Sugar, fat, spices)

Conditioning

Extrusion

Cooling

Packaging

Storage

Figure 3: Flow chart for the production of extruded snacks from BGN, “acha” and carrot composite flour

31

3.4.0 ANALYSIS OF RAW MATERIAL AND PRODUCT Chemical, microbial, physical and sensory analyses were performed on the raw

materials and the products as described below.

3.4.1 Proximate Composition

Proximate composition was determined by using standard method (AOAC, 1995). This

involved the determination of fat, moisture content, crude fibre and protein, ash and

carbohydrate content.

Fat

The Soxhlet extraction method AOAC (1995) was used in determining fat content of the

samples. Each of the sample (2.0g) was weighed out using Mettler- HAS balance, and

put in the extraction thimble and plugged. It was then placed back in the Soxhlet

apparatus. Weighed flat bottom flask (B) was thereafter filled to about three quarters of

its volume with petroleum ether of 40-600C boiling point range. The apparatus was then

set up and the experiment was carried out for a period of 4-8 hours after which

complete extraction was made. The petroleum ether was recovered by evaporation

using water bath (Technicol England) and the remaining sample in the flask was dried

in the oven (Gallenkamp) at 800C for 30 minutes and cooled in a dessicator and finally

weighed using Mettler HAS. The difference in the weight of the empty flask and the

flask with oil gave the oil content, which was calculated as percentage fat content as

follows;

F =C - B x 100 A 1 Where,

A = Weight of sample B = Weight of empty flask C = Weight of flask + oil Protein

The crude protein content of the samples was determined by the semi-micro Kjeldahl

technique described by AOAC (1995). The sample (1.0g) was put into a Kjeldahl flask

and 3.0g of hydrated cupric sulphate (catalyst) were added into the flask. Twenty

mililiters (20ml) anhydrous sodium sulphate and concentrated sulphuric acid (H2SO4)

were added to digest the samples, stoppered and swirled. The flask with its content

was then swirled occasionally until the liquid was clear and free from black or brown

32

colour. The clear solution was then cooled and made up to 100ml with distilled water

and a digest of about 5ml was collected for distillation.

60% sodium hydroxide solution (5ml) was put into the distillation flask and distilled for

some minutes. The ammonia that distilled off was absorbed by boric acid indicator,

which was titrated with 0.1ml hydrochloric acid (HCl). The titre value of the end point,

at which the colour changed from green to pink was taken. The crude portion was

calculated as percentage crude protein thus:

Percentage crude protein = 0.0001410 x 6.25 x 25 x T x 100

W x 5

Where W = weight of sample T = Titre value A factor of 6.25 was used to convert nitrogen to protein.

Soluble protein

Nitrogen solubility was determined according to the method of Mattil (1971). One gram

of sample was mixed with 15ml of water and pH adjusted to 7.0 by the addition of dilute

alkali or acid. The volume was adjusted to 20ml with water and shaken for one hour at

room temperature and centrifuged at 5000rpm for 20 min. The solublized nitrogen was

expressed as per cent of total nitrogen of the sample. A factor of 6.25 was used to

convent soluble N to protein.

Crude Fibre

The crude fibre content of the sample was determined using the method described in

AOAC (1995). Two grams (2.0g) W1, of samples was weighed using Mettler HAS

balance, and put in a 250-ml beaker, and boiled for 30 minutes with 100ml of 0.12ml

H2SO4 and filtered through a funnel. The filtrate was washed with boiling water until the

washing was no longer acidic. The solution was boiled for another 30 minutes with

100ml of 0.012M NaOH solution, filtered with hot water and methylated spirit three

times. The residue was transferred into a crucible and dried in the oven (Gallenkamp)

for 1 hour. The crucible with its content was cooled in a dessicator and then weighed

(w2) using Mettler HAS balance. This was taken to a furnace for ashing at 6000C for 1

hour. The ashed sample was removed from the furnace and put into a dessicator to

33

cool and later weighed (w3) using Mettler, HAS.balance. The percentage crude fibre

was calculated thus:

%Crude fibre = loss of weight on ignition/weight of sample x 100

Ash

The crucibles were washed thoroughly, dried in hot oven (Model: Gallenkamp, size: 3

OV165) at 1000C, cooled in a dessicator and their empty weights were recorded.

Sample weighing 3g each was weighed into the labeled porcelain crucible. Initial

carbonization was conducted by placing the dish over a Bunsen flame and heated

gently until the content turned black. The samples were then subjected to burning in a

muffle furnace at 550oC for 5 hours. The ashed samples were removed from the muffle

furnace, moistened with a few drops of water to expose the unashed carbon, and re-

ashed at 550 oC for another hour. The resulting samples were removed from the

furnace, cooled in a dessicator and weighed soon after reaching room temperature.

The percentage ash was calculated using the expression;

Percentage ash content = 100×−B

AC

Where: A= Weight of empty dish B= Weight of sample in g C= Weight of dish + ash

Carbohydrate Content

The carbohydrate content of each sample was determined by difference. The difference

between 100 and the sum of the percentages of moisture, protein, fat, fibre and ash of

each sample was found, and expressed as percentage carbohydrate.

3.4.2.0 Mineral Content Analysis

Determination of minerals like Ca, P, Na, Mg, K, Fe, Cu and Mn were carried out

according to AOAC (1995). Sample weighing 10g was transferred into a crucible, and

heated over flame to volatilize as much of the organic matter as possible, before

34

transferring into a muffle furnace to burn at 450°C for 5-7hours. To the ash was added

10ml of dilute HCl, boiled for a few minutes, and made up to 100ml with distilled water.

This was used for the mineral analysis as described below.

3.4.2.1 Determination of Phosphorous

Determination of phosphorous was done according to the method of AOAC (1995).

Twenty five grams (25g) of ammonium molybdate and 1.25g of ammonium

metavanadate were added to 300ml of distilled water, warmed to dissolve, cooled and

made up to 500ml with water. Concentrated HCl (215ml) was diluted to 500ml with

water and mixed with ammonium molybdate-ammonium metavanadate reagent.

Phosphorous stock was prepared by dissolving 0.879g of dried phosphorous

dihydrogen orthophosphate (dried at 105oC for one hour) with water and 1ml of conc.

HCl added. It was diluted to 200ml with the first reagent, and 2ml of toluene was added

to give 1mg/ml. The working standard was prepared by measuring 2ml of phosphorous

to 0, 2, 4, 6, 8, and 10ml of standard phosphorous solution into six 200-ml volumetric

flasks and diluted to mark with water.

Each phosphorous standard solution (5ml) was pipetted into a 500-ml graduated flask.

Molybdate mixture (10ml) was added and diluted to the mark with water. It was allowed

to stand for 15 minutes for colour development, and the absorbance measured at

400nm against blank. A calibration curve (see appendix 1) relating absorbance to mg of

phosphorous was used to read the phosphorus content of the sample solution in mg/ml,

and the number of phosphorous equivalent to the absorbance of the sample blank

determined was calculated.

3.4.2.2 Iron Determination

Phenanthroline method as described in AOAC (1995), was used. Phenanthroline

solution was prepared by dissolving 100mg I,10-phenanthroline molybdate in 100ml

distilled water by stirring and heating to 80oC. Hydroxylamine solution was prepared by

dissolving 10g in 100ml of distilled water, while ammonium acetate buffer solution was

prepared by dissolving 250g in 150ml distilled water. 5ml of the digested sample was

added in a test-tube. Then, 3ml of phenanthroline solution and 2ml of HCl was added.

Hydroxylamine solution (1ml) was added to the mixture and boiled in a steam bath at

600oC for 2 minutes. Then, 9ml of ammonium acetate buffer solution was added and

35

diluted to 50ml with water. The absorbance was taken at 510nm. Calibration curve (see

appendix 2) was prepared by pipetting 2, 4, 6, 8, and 10ml standard iron solution into

100ml volumetric flasks to prepare a solution of known concentrations. The curve

obtained was used to read off the value of iron.

3.4.2.3 Determination of Calcium

Titrimetric method was used for calcium determination as described in AOAC (1995).

The test solution (10ml) was added into 250-ml conical flask. Potassium hydroxide

(25ml), water (25ml) and a pinch of calcium indicator were added, and titrated against

Ethylene Diamine Tetra Acetate (EDTA) dissolution salt solution to an end point. The

volume of EDTA is the volume equivalent of calcium in the solution. The value of

calcium was calculated as shown below:

% Ca = 10.1000

100...

××××××

edofsampleuswt

DFofcalciumwtAtEDTAmolTAvolumeofED.

Where EDTA- Ethylene Diamine Tetra Acetate

DF- Dilution factor

3.4.2.4 Determination of Potassium

Determination of potassium was done according to the method of AOAC (1995). The

ashed sample (2ml) was pipetted and transferred into 3 test tubes and 3ml of water

added; 2ml of sodium cobalt nitrite reagent was added, shaken vigorously and allowed

to stand for 45 minutes and centrifuged 15 minutes. The supernatant was drained off

and to the residue was added 2ml of ethanol and shaken vigorously, centrifuged and

the supernatant drained off. The residue was further washed twice with ethanol and

centrifuged respectively. 2ml of water was added to the residue, boiled for 10 minutes

with frequent shaking to dissolve the precipitate, cooled and 1ml of 1% choline

hydrochloride, 1ml of 2% sodium ferricyanide added, and made up to 6ml with water.

The absorbance was taken at 620nm against a blank.

3.4.2.5 Determination of Manganese

Determination of manganese was done according to the method of AOAC (1995).

Ashed sample (2ml) was transferred into 3 test tubes and 3ml of water added; 0.5ml of

conc. sulfuric acid was added and boiled for 1 hour. 0.1g of sodium m-periodate was

36

added and boiled for 10 minutes, cooled and made up to 10ml with water. The

absorbance was taken at 570nm against a blank.

3.4.2.6 Determination of Copper

Determination of copper was done according to the method of AOAC (1995). Ashed

sample (2ml) was transferred into 3 test tubes and 3ml of water added; 1ml of

versanate-citrate solution was added. The mixture was made alkaline with ammonia

and 0.1ml of 1% sodium diethyldithiocarbamate added. 5ml of carbon tetrachloride was

added and shaken vigorously; allowed to separate and the absorbance of the lower

layer taken at 440nm against a blank.

3.4.2.7 Determination of Magnesium

Determination of magnesium was done according to the method of AOAC (1995).

Ashed sample (2ml) sample was transferred into 3 test tubes and 3ml of water added;

2ml of 10% sodium tungstate, 2ml of 0.67N sulfuric acid were added, centrifuged for 5

minutes. 5ml of the supernatant was taken added 1ml water, 1ml of 0.05% titan yellow,

and 1ml of 0.1% gum ghatti. 2ml of 10% sodium hydroxide was added and the

absorbance taken at 520nm against a blank.

3.4.3. In Vitro Protein Digestibility

The in vitro protein digestibility was determined by the method described by Akeson

and Stahman, (1964). Pepsin followed by pancreatin digest was prepared by incubating

100 mg of protein equivalent sample with 1.5 mg pepsin in 15ml of 0.1N HCl at 37oC for

3 hours. After neutralization with 0.2 N NaOH, 4 mg pancreatin in 7.5 ml of phosphate

buffer (pH 8.0) was added. One ml of toluene was added to prevent microbial growth

and the solution was incubated for additional 24 hours at 37oC. The enzyme was

inactivated by the addition of 10 ml 10% trichloro acetic acid (TCA) to precipitate

undigested protein. The volume was made up to 100 ml and centrifuged at 5000 rpm

for 20 min. The protein content of the clear solution was calculated as the percentage of

the total protein solubilised after enzyme hydrolysis.

37

3.4.4 Functional Properties

Water absorption capacity

This was determined by the method of Sosulski (1962). To 1g of flour in a weighed

centrifuged tube 10 ml of water was added and the material suspended in water by

mixing with a thin glass rod and vortexed for 1 minute. After a holding period of 30 min.,

the suspension was centrifuged at 3000 rpm for 25 min. The supernatant was

discarded and the tube kept mouth downwards at an angle of 45o in an oven at 50oC for

25 min, before keeping it in a dessicator and weighing. The difference in two weights

gave the amount of water absorbed by the material. Water absorption capacity was

expressed as the amount of water absorbed by 100 g of material.

Fat absorption capacity

This was determined by the method of Sosulski et al (1976). To 1g of meal, 10ml of

refined groundnut oil was added and the material suspended in oil by mixing with a thin

glass rod and vortexed for 1 min. After 30 min standing, the suspension was

centrifuged at 300 rpm for 25 min. Volume of free oil was measured. Fat absorption

capacity was expressed as the amount of oil (in ml) absorbed by 100g of meal.

Foam Capacity

Two grams of bambara groundnut flour was mixed with 90ml of water in a Blender, and

whipped at low speed for 5 mins and mixture poured into a 250-ml measuring cylinder

and the total volume was recorded after 30 sec. Foam capacity was expressed as %

volume increase (Lawton and Carter, 1971).

Foam stability was determined by measuring the volume of foam at 2, 5, 10, 15, 30, 60

and 120 mins after pouring the mixture into the cylinder. Foam stability was expressed

as foam volume after the lapse of a particular time interval.

3.4.5 Determination of Anti-Nutritional Factors

Trypsin inhibitor activity

Trypsin inhibitor activity was determined according to the method of Kakade et al.

(1974). One gram of the finely ground defatted flour sample was extracted with 50ml of

0.01N NaOH for 3 hours at room temperature and centrifuged at 10,000 rpm for 20min.

The supernatant was used for estimation after addition of 2-5ml distilled water. Aliquots

38

ranging from 0.2 to 1.0 ml were pipetted into duplicate test tubes and volume adjusted

to 2.0 ml with distilled water. To each tube 2.0 ml of trypsin solution was added, placed

in water bath at 39oC and 5 ml of previously warmed (37OC) N-Benzoylarginine-p-

nitroanilide (BAPNA) solution added. Exactly after 10 min, the reaction was terminated

by adding 1.0 ml of acetic acid (30%), and the absorbance measured at 410 nm against

the reagent blank. The reagent blank was prepared by adding 1.0 ml of acetic to test

tubes containing trypsin and water (2.0 ml) followed by addition of 5.0 ml of BAPNA.

The rest of the procedure was same. Sample blanks were prepared using sample

extract. Trypsin inhibitory unit per ml (TIU/ml) vs. volume of extract was plotted and

extrapolated to zero (see appendix 3). One trypsin unit was defined as an increase in

0.01 absorbance units at 410 nm per 10ml of reaction mixture under condition used.

Trypsin inhibitor activity was expressed in terms of trypsin inhibitor units (TIU) and the

value expressed as TIU/mg of sample.

Phytate

The phytate content of the flour was determined by Maga (1982) method. Two (2g)

grams of each finely ground flour sample was soaked in 20ml of 0.2 N HCl and filtered.

After filtration, 0.5 ml of the filtrate was mixed with 1ml ferric ammonium sulphate

solution in a test tube, boiled for 30 min in a water bath, cooled in ice for 15min and

centrifuged at 3000 rpm for 15 min. One millimeter of the supernatant was mixed with

1.5ml of 2,2- pyridine solution and absorbance measured in a Spectrophotometer at

519nm. The concentration of phytic acid was obtained by extrapolation from a standard

curve using standard phytic acid solution. For plotting the standard curve (see Appendix

4) different concentrations (0.2-1.0ml) of sodium phytate solution containing 40-200µg

phytic acid were taken and made to 1.4ml with water (0.4OD corresponding to 70µg

phytic acid).

Tannin

Tannin content was determined by the Folis- Denis calorimetric method described by

Kirk and Sawyer (1998). Five-gram (5g) bambara groundnut sample was dispersed in

50ml of distilled water and shaken. The mixture was allowed to stand for 30min at 28°C

before it was filtered through Whatman No.42 grade filter paper. Sample containing 2ml

extract was dispersed into a 50-ml volumetric flask. Similarly 2ml standard tannin

39

solution (tannic acid) and 2ml of distilled water were put in separate volumetric flask to

serve as standard and reagent was added to each of the flask and the 2.5ml of

saturated Na2CO3 solution added. The content of each flask was made up to 50mls with

distilled water and allowed to incubate at 28ºC for 90min. Their respective absorbance

was measured in a spectrophotometer at 260nm using the reagent blank to calibrate

the instrument at zero.

Haemagglutinin

The method described by Pull et.al. (1978) was used. Fresh blood was collected from a

student (blood group B) using a 5-ml syringe. The red blood cells (erythrocytes) were

extracted from the whole blood suspension in a clinical tube centrifuged at 2000rpm for

10 minutes. One volume of the red blood cells was diluted with 4 volumes of cold 0.9%

saline solution, centrifuged at 2000rpm for 10min and the supernatant fluid discarded.

The sedimented cells were washed with saline in this manner at least three times, until

the supernatant fluid was colourless. The washed red blood cells were introduced to

phosphate buffered saline (0.006M phosphate buffer pH 7.4 in saline) about 4ml of cells

per 100ml of phosphate buffered saline. To 10 parts of this suspension was added 1

part of 2% trypsin solution and the mixture incubated at 37°C for 1hour. The trypsinized

red blood cells were then washed 4 to 5 times with 0.9% saline as earlier described to

remove traces of trypsin. About 2g of bambara groundnut flour was dissolved in 20ml

distilled water, 2ml of the solution was centrifuged at 2000rpm for 10 minutes in order to

obtain a clear solution. The supernatant which was collected and used as crude

agglutinin extract was diluted 2 fold increase to a final dilution of 1:640 in phosphate

buffered saline portions. Each dilution (25ml) was transferred to wells in a microtitre

plate and 25ml volume of 3% suspension of trypsinized type “B” human erythrocytes

was added to each well. The titres were recorded after 3hours at room temperature.

Trypsinized type “B” human erythrocyte was prepared by treatment of a 3% (v/w)

suspension of cells in phosphate-buffered saline for 1hour with Sigma type ix porcine

pancreatic trypsin. One haemagglutinin unit (HU) - defined as the least amount of

haemagglutinin that will produce evidence of agglutination of 25FI of a 3% suspension

of washed, trypsinized type “B” human erythrocytes after 3 hours incubation at room

temperature. Da x Db x S

Hu/g= --------------------------------- V

40

Where V= volume of extract in tube; Da= Dilution factor of extract in tube 1; Db= Dilution factor of tube containing 1Hu; S = ml original extract/gcal

3.4.6.0 Determination of Vitamins

Determination of vitamin A - Biological method for assessment of vitamin A in form of β-

carotene status was used (IVACG, 1982), while determination of vitamins B1, B2,

B3 and

B6 were carried out using Snell and Snell (1953) methods.

3.4.6.1 ββββ-carotene and Vitamin A

Sample weighing 1gm each was macerated with 100ml of distilled water, and 2ml was

pipetted in duplicate into a glass stoppered test tube. An equal volume (2ml) of ethanol

was added drop wise with mixing to give 50% solution (v/v). At this concentration the

protein precipitated and free from retinol and retinyl esters was extracted by addition of

3ml hexane. The tube was stoppered and the contents mixed rigorously on the vortex

for 2 minutes to ensure complete extraction of carotene for 5 – 10 minutes at 600 -1000

x g to obtain a clean separation of phases. Two (2)ml of the upper hexane extract was

pipetted. Absorbance due to carotenoids at 450nm was read against a hexane blank

(A450).

After determining A450, the cuvettes were removed and the hexane was evaporated to

dryness under a gentle stream of nitrogen at 40 – 50oC water bath, while avoiding

splashing on the test tube wall. At the point of dryness during evaporation, the residue

was immediately re-dissolved and rehydrated by addition of 0.01ml of a mixture of

chloroform – acetic anhydride (1.1v/v). The cuvettes or tubes were cupped to minimize

evaporation and was protected from light to avoid oxidation.

The spectrophotometer at 620nm was preset consisting of 0.1ml chloroform –acetic

anhydride mixture and 1.0ml of trichloro acetic acid (TCA) – chloroform chromagen

reagent, 1.0ml of trifluro acetic acid (TFA) could also be used. The cuvette containing

the sample was placed in the spectrophotometer and 1.0 TCA chromagen reagent

added to the cuvete from a rapid delivery pipette. These steps were carried out rapidly

and with care since the blue colour fades quickly and chromagen reagent is highly

corrosive. Absorbance reading was recorded (A620) at exactly 15 seconds (t15) and at

30 seconds (t30) after addition of reagent. A standard curve (see Apendix 5) was plotted

from the A620 values on ordinary rectangular coordinate paper where the ordinate was

41

the A620 value and the abscissa was the µg vitamin A/tube and a factor (FA620)

calculated as shown below:

FA620= 620

/min

A

tubeAvitagµ

Vitamin A was calculated using this formular

Total carotenoids (as β-carotene/dl) = A620 x FC450 x 150

Where, FC450 = constant determined in the laboratory

150 = dilution factor

Likewise vitamin A was calculated (as µg retinol/dl) = A620 x FA620 x 75

Where F A620 = 1/slope of standard curve=1/0.0228=35.7

3.4.6.2 Vitamin B 1 (Thiamin)

The content of Vitamin B1 in the samples was determined according to the Colorimetric

method (Snell and Snell, 1953), of Analysis. Thiamine hydrochloride (50mg) was

weighed using Mettler-HAS balance into a 100-ml volumetric flask, and dissolved in

100ml of water. Sample weighing 1gm each was macerated with 100ml of distilled

water. Each sample solution (2ml) was pipetted separately into 100-ml separation

funnels, 2ml reagent solution was added, and mixed for 1minute, before allowing it to

separate. Isobutyl alcohol (15ml) was added, the mixture was shaken for 2 minutes,

moving the funnels up and down to separate the isobutyl alcohol layer. It was dried by

passing through anhydrous sodium sulphate, then the absorbance was determined at

367nm (Spectro 21D PEC Medicals USA) using isobutyl alcohol as blank. A standard

curve (see Appendix 6) was plotted and the slope used in calculating vitamin B1 as

shown below:

Vitamin B1 = (A x DF)/slope of std. Curve

Where, A – Absorbance

DF- Dilution factor =50

3.4.6.3 Vitamin B 2 (Riboflavin)

The Vitamin B2 content of the snacks was determined using Colorimetric method (Snell

and Snell, 1953). Riboflavin (6mg) was weighed into a 100-ml volumetric flask and

made up to mark with distilled water. The solution (20ml) was diluted in 100ml of

distilled water. The solution was pipetted separately into 2 volumetric flasks (25-ml).

42

Denigee’s reagent solution (5ml) was diluted in 100ml of water. Five (5ml) of each

standard solution was pipetted separately into 2 volumetric flasks (25ml) and 5ml of

denigee’s reagent was added. The flasks were shaken and allowed to stand for 15

minutes before reading their absorbance in 525nm (Spectro 21D PEC Medicals USA)

against a blank solution of 10ml and 5ml of the denigees reagent. A standard curve

(see Appendix 7) was plotted, and the slope used to calculate vitamin B2 as shown

below:

Vitamin B2 = (A x DF)/slope of std. curve

Where, A – Absorbance

DF- Dilution factor =5

3.4.6.4 Niacin

The colometric method (Snell and Snell, 1953) was used to determine niacin content of

the samples. Volumetric flask (100-ml) was weighed using Mettler, HAS balance and

50mg of niacin amide added, dissolved and made up to mark with distilled water. Ten

(10) ml of the solution was put into another 100-ml volumetric flask and made up to

mark with distilled water.

The standard (5ml) was measured into a test tube. Separately 2ml of ammonia buffer

solution and 1ml of distilled water were added, after which 1ml of 2, 6 dichloroquinone

chlorimide solution was added. The solution was shaken vigorously and the

absorbance was taken immediately at 60 to 80nm against a blank.

AT x WS x 3.3 x 100 x 25 x 5 x 5 = mg/5ml AS 100 100 TV 5 25

Where AT = Absorbance of test solution AS = Absorbance of standard solution WS = Weight of standard taken VT = Volume of syrup taken

3.4.6.5 Vitamin C The direct calorimetric method as described by Kalia (2002) was used to determine the

vitamin C content of the samples. It was based on the extent to which a 2,6-

43

dichlorophenol-indephenol solution is decolourized by ascorbic acid in sample extracts

and in standard ascorbic acid. The sample (50g) was blended with an equal weight of

6% HPO3, and an aliquot of the macerate was made up to 100ml.

Plotting of standard curve : The requisite volume of standard ascorbic acid solution-1,

2, 2.5, 4 and 5ml- was pipetted into dry cuvettes, and made up to 5ml with 2% HPO3.

Dye solution (10ml) was added with a rapid delivery pipette and the cuvette shaken

prior to taking the reading within 15-20 sec. The Calorimeter was set to 100%

transmission using a blank consisting of 5ml of 2% HPO3 solution and 10ml of distilled

water. The absorbance of the red colour of the standard solution was measured at

518nm, and absorbance plotted against concentration (see Appendix 8).

The sample extract (5ml) was placed in a cuvette, 10ml of dye was added and

the reading was taken as in standard. The concentration of ascorbic acid was read from

the standard curve and used to calculate the ascorbic acid content as shown below:

(mg of ascorbic acid per 100g of sample)

Ascorbic acid content x Vol. made up x 100 =-------------------------------------------------------------------------------- ml of sol. taken for estimation x 1000 x wt. of sample taken

3.4.7.0 Experimental design/data analysis

The experiment was designed using Completely Randomized Design (CRD). SPSS

version 13 was used to analyse the data obtained statistically using analysis of variance

(ANOVA), and mean separation was done by Duncan(1955) multiple range test at 5%

level of probability.

44

CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 Effect of Processing Treatments on the Proximat e Composition of Bambara Groundnut

The result of proximate composition of bambara groundnut (BGN) and effect of different

processing treatments like roasting, germination and a combination of both is presented

in Table 5. The moisture (10.30%), crude protein (21.85%), fat (6.90%), ash (3.64%),

fibre (3.42%) and carbohydrate (53.93%) of the raw bambara were comparable to

values reported by Poulter (1981), and Obizoba and Egbuna (1992). No significant

changes were observed in protein, fat and ash content of the germinated BGN flour.

However, roasting on the other hand brought about significant reduction in the moisture

content. The slight increase in the protein (21.85% to 23.09%) and fat (6.9% to 7.33%)

contents noticed in the roasted flour could be probably due to concentration of nutrients

as a result of moisture lost during roasting (FAO, 1982).

Table 5: Effect of Processing Method on the Composi tion of Bambara Groundnut (BGN)

Sample Code

Moisture (%)

Protein (%) Fat (%) Ash (%) Fibre (%) *Carbohydrate (%)

Energy (KCal)

BGN 10.30a±0.91 21.85b±1.34 6.90a±1.02 3.64a±0.98 3.42a±0.76 53.89b±1.30 365.06 RBGN 7.01b±0.73 23.09a±0.99 7.33a±1.25 3.76b±0.70 3.21a±1.08 55.60b±1.50 380.73 GBGN 10.79a±0.67 22.18b±0.54 6.73a±0.54 3.58a±0.50 3.60a±0.79 53.12b±0.86 361.57 G/RBGN 7.24b±0.79 23.20a±1.80 7.16a±0.48 3.85a±1.11 3.52a±0.90 55.03ab±0.16 377.36

Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05). *Carbohydrate calculated by difference. SD= Standard deviation BGN = Raw Bambara Groundnut; RBGN = Roasted Bambara Groundnut GBGN= Germinated Bambara Groundnut; G/RBGN =Germinated/Roasted Bambara Groundnut

4.2 Effect of Processing Method on Some Anti-Nutrit ional Factors Table 6 shows the effect of processing method on some anti-nutritional factors. Just like

other legumes, bambara nut contains anti-nutritional factors such as trypsin inhibitor,

tannins (polyphenols), phytate, haemagglutinins. Processing methods such as

dehulling, milling, soaking, cooking, germination, fermentation, autoclaving/roasting and

frying have been found to reduce or eliminate these anti-nutritional factors (FAO, 1982).

The result of this experiment is in agreement with this observation. The processing

treatments given had significant (p<0.05) effect in reducing the anti-nutritional factors.

Roasting reduced the concentration of trypsin inhibitor by 37.5% (from 17.94mg/g to

45

11.21mg/g), germination reduced it by 17% (from 17.94mg/g in the raw bambara to

14.85mg/g) while a combination of germination and roasting brought the highest

reduction of 68% (from 17.94mg/g to 8.73mg/g). The nutritional implication of these

reductions in the concentration of trypsin inhibitor is that it will lead to improvement in

protein and digestibility. Negi et al. (2001) and Archana et al. (2001) reported higher

protein digestibility/quality after heat treatment (autoclaving and roasting), which they

attributed to destruction of heat labile protease inhibitor and opening up of protein

structures by denaturation, leading to increased accessibility of the protein to enzymatic

attack. Germination/malting has also been reported to improve protein digestibility

(Nnanna and Philips, 1990). Increased protein quality/digestibility upon germination

may be probably due to decrease in the anti-nutritional factors like tannin, phytate and

trypsin inhibitors, modification and degradation of storage proteins by the action of

proteolytic enzymes. Tannins are generally defined as soluble astringent, complex and

phenolic substances of plant origin, which play significant role in the reduction of dietary

protein digestibility by complexing either with dietary protein or digestive enzyme.

Table 6: Effect of Processing Methods on Some Anti- Nutritional Factors in Bambara Groundnut

Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05). SD= Standard deviation Tannin content was significantly reduced by the roasting process to 56.5% (from

0.62mg/100g in the raw sample to 0.35mg/100g). Germination reduced tannin by 21%,

while a combination of roasting and germination caused 55% reduction of tannin

content in the sample (from 0.62 to 0.28mg/100g). This reduction brought about

improvement in nutritional quality. Phytate level reduced from the initial content of 100%

(255mg/100g) in raw bambara to 69.8% (178mg/100g) in roasted sample, 75.3%

(192mg/100g) in germinated sample and 47% (120mg/100g) in the germinated and

roasted samples. The Haemagglutinin activity was also reduced from 100%

Processing Treatments on Bambara-Nut

Trypsin Inhibitor Units

(TIU) mg/g

Tannic acid (equivalent)

mg/100g

Phytate (mg/100g)

Haemagglutinin (Hu/mg protein)

Raw 17.94a±0.58 0.62a±0.89 255d±0.36 6.50a±0.78 Roasted 11.21c±1.23 0.35c±1.01 178b±1.42 3.80c±65 Germinated 14.85b±0.69 0.49b±0.22 192c±0.98 5.20 b±1.32 Germinated & Roasted

8.73d±1.24 0.28d±0.05 120a±0.73 2.10d±0.96

46

(6.50Hu/mg/protein) in raw bambara flour to 58.5% (3.80Hu/mg/protein) in roasted

sample, 80% (5.20Hu/mg/protein) in germinated sample to 32.3% (2.10Hu/mg/protein)

in germinated and roasted sample. Reduction in trypsin inhibitor, tannin content, phytic

acid, and haemagglutinin contents of some tropical legumes processed by cooking,

autoclaving and roasting had been reported by Igbedioh et al. (1994) and Apata and

Ologhobo (1994). Similarly Obizoba and Egbuna (1992) reported reduction in

haemagglutinin content of germinated bambara groundnut. Phytate reduction by 38 –

46% after germination had been reported by Archana and Kawata (1998) and Salunkhe

et al. (1985). The reduction was attributed to leaching of phytate ions and increased

activity of phytase enzyme during germination.

4.3 Effect of Processing Methods on the Functional Properties of Bambara Groundnut

The effect of processing methods on the functional properties of Bambara groundnut is

presented in Table 7.

Table 7: Effect of Processing Methods on Selected F unctional Properties of Bambara Groundnut Treatments Water

binding capacity

(g/g)

Oil binding capacity

(g/g)

Foaming capacity (vol/ml)

Nitrogen solubility (%)

Raw 1.7b±0.63 1.3a±0.42 33.3c±0.25 85.40b±0.66 Roasted 1.6b±0.70 1.3a±0.01 24.0a±0.06 46.80d±0.73 Germinated 1.0a±0.29 1.1a±0.83 41.7d±0.41 92.10a±1.32 Germinated /Roasted

1.2a±0.44 1.2a±0.92 28.9b±0.11 49.20c±0.95

Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05). SD = standard deviation

The functional properties of food proteins determine their behaviour in food systems

during processing, storage, preparation and consumption. These functional properties

and the interaction of proteins with other components directly and indirectly affect

processing applications, food quality and ultimate acceptance. Germination process

was found to significantly (p< 0.05) affect the water binding capacity of bambara flour

(see Table 7). Roasting on the other hand did not have any significant (p>0.05) effect on

the water absorption capacity. Padmashree et al. (1987) reported that polar amino

acids of proteins had an affinity for water and denatured proteins bind less water. Fat

binding capacity has been attributed to the physical entrapment of oil. This is important

47

since fat improves taste, texture and mouth feel (Kinsella, 1976). Processing methods

did not have significant (p>0.05) effect on the fat binding capacity 1.3g/g (raw), 1.3g/g

(roasted), 1.1g/g (germinated) and 1.2g/g for (germinated/roasted). Roasting

significantly (p<0.05) reduced the foaming capacity by 28%, while germination

significantly (p<0.05) increased it by 25.2%. Nitrogen solubility profile over a range of

pH is used as a guide to protein functionality since this relates directly to many

important properties of protein (Lin et al.1974; Sosulki et al. 1976; Mc Waters and

Holms, 1979). Roasting reduced Nitrogen solubility of bambara flour from 85.40%

solubility in the raw samples to 46.80% in the roasted flour, and 49.20% in the

germinated and roasted flour. Padmashree et al. (1987), observed that heat treatment

(roasting, puffing, boiling and pressure cooking) significantly(p<0.05) reduced protein

solubility in the pH range of 2 – 10. Reduction in Nitrogen solubility with heat processing

had also been reported for sunflower, rapeseed, groundnut and soyabean

(Padmashree et al. 1987). However, there seem to be slight increase in the solubility

with germination, as the protein solubility increased from 85.40% in the raw flour to

92.10% in the germinated flour.

4.4 Effect of Treatment Methods on the Consume r Acceptability of the Extruded Snacks from Treated Bambara Groundnut .

The effect of treatment methods (roasting, germination, roasting & germination) on the

consumer acceptability of the extrudates is presented in Table 8. There were

significant differences (p< 0.05) among the extrudates in colour, taste, flavour and

overall acceptability, while there were no significant difference (p> 0.05) among them in

texture and mouthfeel. The extrudates from roasted bambara groundnut had

significantly (p<0.05) higher mean score in colour, taste, flavour and overall

acceptability. This relatively higher mean score of the roasted samples could be

probably due to the roasted flavour and aroma imparted on the bambara groundnut

during roasting. Controlled roasting of nuts brings about development of desirable

roasted aroma in foods, which are described as nutty, burnt and coffee like, due to the

formation of pyrazine compounds that also reflects the extent of browning colour

development in the product (Powrie and Nakai, 1981). The relatively lower mean

sensory scores recorded in the germinated extrudates could be probably due to the

slight bitter after taste observed in germinated samples.

48

Despite the low mean scores, the samples were not however rejected. Roasting was

found to improve the organoleptic qualities of the extrudates, and hence it was

subsequently adopted for formulation of the blends with acha and graded levels of

carrot.

Table 8: Consumer Acceptability Study of the Extrud ates from Treated Bambara Groundnut

Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (P< 0.05). *BGN-Bambara Groundnut. SD = standard deviation 4.5. Proximate Composition of Hungry Rice “Acha” an d Fresh Carrot

The chemical composition of carrot used as a source of ß-carotene is presented in

Table 9. Carrot contains (for each 100g) 9.80g carbohydrate, 0.9g protein, 0.10g fat,

1.9 fibre, very rich in ß-carotene (840.8mg/100 retinol) 0.80mg iron, 38.3mg Ca, 2.96mg

Zinc. It also contains a good source of vitamin B1, B2, B6, vitamins C and naicin. The

composition of carrot in this study compares with the values reported by Chan (2007).

Table 9: Chemical Composition of “Acha” and Carrot (per 100g of sample) Parameters Acha Carrot Moisture, % 10.92±0.95 87.50±2.34 Protein, % 9.85±0.56 0.90±0.12 Ash, % 1.21±0.04 0.80±0.20 Fat, % 1.34±0.05 0.10±0.01 Fibre, % 2.24±0.6 1.90±0.15 Carbohydrate, % 74.44±2.21 9.80±1.13 Energy (Kcal) 349.22±5.55 42.60±2.33 Ca (mg) 64.2±3.21 38.34±1.56 Potassium (mg) ND 328.0±2.78 Magnesium (mg) 4.51±0.93 15.40±0.98 Zinc (mg) ND 2.986±0.45 Iron (mg) 23.01± 1.11 0.80±0.11 Vitamin A in form of ß-carotene (In mg/100 retinol) ND 840.8±6.32 Vitamin C (mg) 28.60±2.01 10.7±1.05 Vitamin B1 (mg) 0.10±0.01 0.06±0.01 Vitamin B2 (mg) 0.05±0.02 0.05±0.02 Vitamin B3 (mg) 2.00±0.37 0.96±0.12 Vitamin B6 (mg) ND 0.04±0.01

Values are mean of triplicate determination ±SD. ND – Not determined

Treatments Colour Taste Texture Flavour Mouthfeel Overall Acceptability

Raw BGN* 6.6±0.96b 6.8±0.71ab 7.1±0.19a 6.8±0.86ab 7.0±0.52a 6.8±0.92b Roasted BGN* 7.5±1.01a 7.7±0.86a 7.3±0.90a 7.6±0.66a 7.4±0.45a 7.7±0.50a Germinated BGN* 6.5±0.73b 6.0±0.61b 7.2±0.30a 6.5±0.25b 7.1±0.84a 6.3±0.80b Roasted/Germinated 7.0±0.20ab 6.7±1.14a 7.2±0.79a 6.9±0.60ab 7.3±0.88a 6.6±0.38b

49

The chemical and energy composition of “acha” revealed 10.92% moisture, 9.85%

protein, 1.21% fat, 1.34% ash, 2.24% fibre, 74.44% carbohydrate, and 349.22Kcal of

energy per 100g (dry weight basis). It also contains appreciable quantities of

micronutrients such as Ca, Mg, Fe and vitamins (B1, B2, B3 and C). These values

compare with those reported by Nnam (2000) and Ayo et al. (2007).

4.6 Effect of Extrusion on the Proximate Compositio n of Bambara/”Acha” and Carrot flour Blend

The proximate composition of blend of bambara/”acha” and carrot flour before, and

after extrusion is presented in Table 10. Expectedly, extrusion cooking reduced the

moisture content from 10.52% in the bambara/acha blend to 4.05% in the extruded

sample. Equally, the moisture content of the 5 – 20% carrot fortified blends reduced

from 9.61 – 10.06% in the blends to 4.05 – 4.61% in the extruded products. The

moisture reduction could be probably due to the loss of moisture on extrusion as a

result of high temperature in the extruder and subsequent drying of the extruded

products. There was also a slight increase in the protein and ash contents after

extrusion. These could be probably due to concentration of these nutrients, as a result

of moisture loss.

Table 10: Proximate Composition of Un-extruded & Ex truded Bambara- Acha Containing Graded Levels of C arrot

Sample Code

Moisture (%) Protein (%) Fat (%) Ash (%) Fibre (%) Carbohydrate (%)*

Energy (KCal)

BEFORE EXTRUSION Before BAB

10.12a±0.93

15.19b±0.31

3.24a±0.32

2.73a±0.65

1.96b±0.44

66.76b±0.22

356.96

BAC5 10.06a±0.74 14.68bc±0.90 3.03a±0.7c 2.90a±0.85 2.02b±0.81 67.31b±0.65 355.23 BAC10 9.8a±0.85 14.11c±0.18 2.87a±0.45 2.82a±0.77 2.69a±1.08 67.71b±0.88 353.11 BAC15 9.9a±0.12 13.61cd±1.05 2.92a±0.32 2.89a±0.15 2.95a±0.92 67.73b±0.34 351.56 BAC20 9.61a±0.11 13.09d±0.99 2.48a±0.83 2.68a±0.07 3.03a0.21 69.11c±0.17 3501.12

AFTER EXTRUSION After BAB

4.05b±0.74

16.25a±0.65

6.80b±0.08

3.47ab±0.82

1.89b±201

67.54b±1.08

396.88

BAC5 4.23b±0.49 15.99a±0.38 6.31b±0.08 3.83ab±0.82 2.34b±0.25 67.30b±0.21 388.71 BAC10 4.61b±0.32 15.32ab±0.18 6.39b±0.63 3.78ab±1.62 2.78a±0.71 67.12b±0.98 387.27 BAC15 4.00b±0.91 15.15b±1.08 6.73b±0.12 3.99b±0.99 2.90a±1.04 67.23b±0.35 390.09 BAC20 4.18b±0.78 15.02b±0.77 6.46b±0.96 3.93b±1.18 3.06a±0.21 67.35b±0.67 387.22

CEP 4.10b±0.33 6.00e±055 1.00c±0.25 1.80c±0.67 1.00c±0.33 86.10a±0.88 383.4 Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05). *Calculation by difference BAB=Roasted bambara/Acha blend without carrot (control); BAC5=Roasted bambara/Acha blend with 5% Carrot; BAC10=Roasted BGN/Acha with 10% Carrot; BAC15=Roasted bambara/Acha with 15% Carrot; BAC20=Roasted bambara/Acha with 20% Carrot; CEP= Commercial Extruded Product

50

However, the bambara/acha blend with zero carrot fortification (BAB) as well as its

extrudate had significantly higher protein content than the 15 and 20% carrot fortified

samples. This could probably due to the fact that replacement of the bambara

groundnut/”acha” blend with carrot gradually reduced the protein content of the

resultant composite flour, because bambara groundnut naturally contains higher

proportion of protein than carrot. The commercial extruded snack sold in the market

was found to contain 6% protein, which was less than the protein content of the

extruded snacks in this study.

The BGN based blends had relatively high protein content that reflected in the extruded

snacks, making them nutrient dense food products that would be children friendly.

According to US Committee on Dietary Allowance (1980), children between 5 and 14

years old require 34 – 46g of protein/day for body building, repairs, metabolism and

proper functioning of the body for better health benefit. These extruded snacks would

provide between 30 and 50% Recommended Dietary Allowance (RDA) for protein

requirements of 5-14 years old school age child, if 100g of it is consumed daily. The

Protein content of snack containing 5% added carrot flour was 15.99%, which satisfies

about 48% RDA protein requirement of a child if 100g of it is consumed. Snacks with

10% carrot inclusion had 15.32% of protein (which satisfies 44.5% RDA), 15% carrot

containing samples had 15.15% protein content (which satisfies 43% RDA) and 20%

carrot added samples had 15.02% protein content (which satisfies also about 43%

RDA).

There was significant (p<0.05) increase in the fat content of the blends from a range of

2.48% – 3.34% to 6.06% – 6.80% in the extruded products. This increase could be

attributed to the vegetable fat added to the formulation. Also noticeable was a slight

increase in the fibre with increasing addition of carrot in the blends, and this follows the

same trend after extrusion. The fibre content of the product satisfies 40 – 69% of a

child’s daily RDA (5g of dietary fibre). The caloric value of the extruded product is

significantly (p<0.05) higher than that of the blends, which rose from 351.12 –

356.96Kcal in the blends to 387.27 – 396.88Kcal in the extruded samples. These

increases could be probably due to the increase in the fat content as a result of the

added vegetable oil. The high caloric values will provide adequate energy for children

who need a lot of it for their daily activities.

51

4.7 Sensory Qualities of Extruded Snacks

The sensory properties of the extruded snacks from BGN/”Acha” fortified with carrot

(BAC5 – BAC20) and the control sample without carrot (BAB) are presented in Table

11. Carrot addition was found to decrease the ratings of all the sensory attributes

(colour/appearance, taste, mouthfeel, flavour, crunchiness/texture and overall

acceptability) analysed. The extruded snack with 0% carrot inclusion was the most

acceptable followed by 5% carrot addition, 10% carrot addition, 15% and 20% carrot

addition. The lower sensory mean score/acceptability of the carrot-fortified extrudates

may be probably because people are not used to this type of product. The carrot taste,

colour and composition may also have affected some of the organoleptic properties,

and may have contributed to the formation of new flavours in the products. However,

there was no significant difference (p>0.05) between the control sample (BAB) and

samples with 5 and 10% carrot in all the sensory attributes analysed, therefore they

were further used for the storage studies.

Table 11: Mean Sensory Scores of Bambara-Acha Extru ded Snacks Containing Graded Levels of Carrots

Sample Code

Colour/ Appearance

Taste Mouthfeel Flavour/ Aroma

Crunch -iness

Texture Overall Acceptability

BAB 7.5a±0.130 7.4a±0.85 7.0a±0.98 7.1a±1.06 7.6a±1.04 7.0a±0.71 7.4a±1.27 BAC5 7.3a±0.71 7.5a±1.34 7.1a±0.39 7.3a±0.73 7.4a±0.85 7.3a±0.92 7.3a±0.99 BAC10 7.4a±0.99 7.19b±0.55 6.5ab±0.54 7.0a±0.84 7.0ab±0.76 7.0ab±0.22 7.0ab±1.26 BAC15 7.0ab±1.01 6.9ab±0.71 6.0b±0.89 6.8ab±1.14 6.9ab±1.91 6.2b±0.35 6.2bc±1.10 BAC20 6.2b±0.46 6.4b±0.84 6.1b±1.17 6.0b±1.47 6.3b±0.77 6.0b±0.41 5.6c±0.53

Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05). SD = standard deviation BAB=Roasted bambara/”acha” blend without carrot (control); BAC5=Roasted bambara/”acha blend with 5% Carrot; BAC10=Roasted BGN/”acha” with 10% Carrot; BAC15=Roasted bambara/”acha” with 15% Carrot; BAC20=Roasted bambara/”acha” with 20% Carrot;

4.8 Effect of Extrusion on the Residual Anti-Nutrie nts

Table 12 shows the level of residual anti-nutrients in the extruded snacks. Extrusion

had significant (P<0.05) effect on the anti-nutritional properties, particularly on trypsin

inhibitor and haemagglutinin. This effect is attributed to moist heating occasioned by

flour hydration and extruder temperature and pressure conditions. Trypsin inhibitor

activities are known to be protease inhibitor, and is destroyed by heat, the extent of the

52

destruction depends on the process temperature, duration of heating, particle size,

moisture content, buffer, pH and screw speed (Pham and Del Rosario, 1987).

Table 12: Effect of Extrusion on Anti-Nutrient Cont ent of BGN/Acha Blends Containing Graded Levels of Carrot

*Not Detected. Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05). BAB=Roasted bambara/”acha” blend without carrot (control); BAC5=Roasted bambara/”acha” blend with 5% Carrot; BAC10=Roasted BGN/”acha” with 10% Carrot; BAC15=Roasted bambara/”acha” with 15% Carrot; BAC20=Roasted bambara/”acha” with 20% Carrot;

In this study, trypsin inhibitor activity and haemagglutinin were not detected, while the

phytate level was reduced to 81.11mg/100g from 91.01mg in the raw blend and to

30.58 from 36.75mg/100g in the extruded samples. Similarly, the tannin content

decreased from 0.16 – 0.26mg/100g to 0.06 – 0.09 mg/100g. This result agreed closely

with the values reported by Nwabueze (2006) for extruded breadfruit snacks.

4.9 Effect of Extrusion on In-Vitro Protein Digesti bility

The effect of extrusion on in-vitro protein digestibility of the BGN/”acha” blends with

added carrot is presented in Table 13. According to FAO/WHO/UNU (1985), protein

nutritional value is dependent on the quantity, digestibility and availability of essential

amino acids, while digestibility is considered as the most important determinant of

protein quality. From the result, there was increase in the invitro-protein digestibility of

the samples after extrusion. Roasted bambara/”acha” blend without carrot (control-

BAB) was found to increase from 84.62% to 86.74%, BAC5 increased from 86.73-

89.05%, BAC10 increased from 86.92-88.62%, BAC15 (87.01-89.08), while BAC20

increased from 86.57 to 89.89%. The increase in the protein digestibility after extrusion

Sample Code

Trypsin Inhibitor Uni ts (TIU) mg/g

Tannin (Tannic acid equivalent mg/100g)

Phytate (mg/100g)

Haemagglutinin (HU/mg protein)

Before Extrusion BAB 8.33a±0.93 0.26a±6.54 91.01a±1.08 5.12 BAC5 7.94a±0.40 0.19b±1.01 86.29b±0.94 4.85 BAC10 7.01b±1.26 0.16b±0.77 81.11c±0.84 4.01 BAC15 6.81b±0.79 0.19b±0.91 83.31c±0.66 4.35 BAC20 6.87b±0.85 0.18b±0.26 85.73b±1.54 4.30

After Extrusion BAB ND* 0.06c±0.03 34.01e±0.11 ND* BAC5 ND 0.09c±0.69 30.58d±0.83 ND BAC10 ND 0.09c±0.12 32.14d±0.25 ND BAC15 ND 0.08c±0.01 36.75e±0.90 ND BAC20 ND 0.08c±0.36 33.68d±0.29 ND

53

might be due to denaturation of proteins, as well as inactivation of enzyme inhibitors

present in raw plant foods, which expose new sites for enzyme attack (Colonna et al.

1989; Nwabueze 2006; Singh et al. 2007). Similar finding had been reported by

Hakansson et al. (1987), Colonna et al. (1989) and Areas (1992), who observed that

the nutritional value in vegetable protein is usually enhanced by mild extrusion cooking

conditions, owing to an increase in digestibility.

Table 13: Effect of Processing on In-vitro Protein Digestibility of BGN- Based Extruded Snacks Containing G raded Levels of Carrot

Sample Code

In-vitro protein Digestibility (%) Before Extrusion After Extrusion

BAB 84.62a±0.52 86.74b±0.95 BAC5 86.73b±0.90 89.05cd±1.52 BAC10 86.92b±0.74 88.62c±b0.84 BAC15 87.01b±0.12 89.08cd±1.01 BAC20 86.57b±0.66 89.86d±0.72

Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05). SD = standard deviation BAB=Roasted bambara/Acha blend without carrot (control); BAC5=Roasted bambara/Acha blend with 5% Carrot; BAC10=Roasted BGN/Acha with 10% Carrot; BAC15=Roasted bambara/Acha with 15% Carrot; BAC20=Roasted bambara/Acha with 20% Carrot;

4.10 Effect of Extrusion on the Mineral Content of the BGN/Acha Blends with added Carrot.

Table 14 shows the effect of extrusion on the mineral content of the BGN/”Acha” blends

with carrot. There was a slight increase in the mineral contents of the blends on

extrusion. The extrudates had slightly higher mineral content than the flour blends,

which was more noticeable in the calcium, phosphorus, potassium, magnesium and

manganese contents. The increase in the mineral content after extrusion also follows

the same trend with the level of carrot incorporation as in the blends before extrusion.

The slight increase in the mineral composition of the extrudates could be probably due

to concentration of nutrients as a result of moisture loss during extrusion. Extrusion

cooking had been found to improve the absorption of minerals by reducing other factors

that inhibit its absorption, but does not significantly (p>0.05) affect mineral composition

of pea kidney bean seeds (Alonso et al. 2001).

55

Table 14: Effect of Processing on the Mineral Conte nt of Extruded Snacks Macro-Minerals (mg/100g) Micro-Minerals (mg/100g)

Sample code Ca P Na K Mg Zn Fe Cu Mn

BEFORE EXTRUSION

BAB 73.92a±2.01

161.33e±1.04

2.08a±0.60

1239.75a±1.09

83.89a±1.80

3.18a±1.03

7.81a±0.90

0.42a±0.38

5.60a±0.44

BAC5 76.85b±0.95 159.94d±1.36 3.61b±1.05 1241.08±1.55 82.68a±0.39 3.42a±0.73 7.58a±1.31 0.35a±0.52 5.03b0.89

BAC10 80.04bc±1.09 155.95c±0.96 3.75b±0.84 1244.17bc±0.68 82.36ab±1.10 3.68ab±0.62 7.35±1.08 0.31a±0.47 4.96b±0.25

BAC15 83.19c±0.98 153.27b±0.78 3.69b±1.31 1246.34c±1.00 82.04ab±0.94 3.89b±1.01 7.12bc±0.74 0.36a±0.59 4.30c±0.12

BAC20 85.98c±0.77 150.52a±0.99 3.84b±1.08 1249.01d±0.88 81.72b±1.21 4.05b±0.78 6.90c±0.52 0.30a±1.01 4.01c±0.63

AFTER EXTRUSION

BAB 78.70bc±0.91 181.05i±0.38 2.32a±0.88 1338.40e±1.10 94.16c±0.44 3.95b±1.72 7.95a±1.03 0.40a±0.21 5.80a±0.24

BAC5 81.63bc±0.67 178.98h±0.40 3.56b±1.05 1330.73f±0.71 93.81c±0.97 4.17b±0.95 7.87a±0.78 0.39a±1.05 5.66a±0.38

BAC10 84.56c±1.18 173.29g±0.72 3.85b±0.23 1364.02g±0.56 93.64c±0.82 4.36bc±0.74 7.69a±1.09 0.39a±0.79 5.35ab±0.11

BAC15 87.89cd±2.03 172.93g±0.72 3.80b±0.48 1369.87h±0.85 93.03cd±0.91 4.04b±0.63 7.26ab±0.45 0.40±1.30 5.01b±0.96

BAC20 90.22d±0.70 170.01f±0.12 3.97b±1.26 1392.561±1.00 92.88d±0.60 4.61c±0.51 7.20ab±0.61 0.33±0.82 5.23b±0.69

*RDA for children 4-10 yrs 800 800 2-5 4-6 250 10 10 50-250mg 2.5-3.8

*% RDA Met 10-12 18-23 78-100 40-100 33-38 32-46 69-79 Above 50 Above 50

. Column means with different superscripts are significantly different at 5% probability level (P< 0.05). Values are mean ± SD of triplicate determination. SD = standard deviation. RDA-Recommended Dietary Allowance (*Source: US Committee on Dietary Allowances (1980)) BAB=Roasted bambara/”acha” blend without carrot (control); BAC5=Roasted bambara/”acha” blend with 5% Carrot; BAC10=Roasted BGN/”acha” with 10% Carrot; BAC15=Roasted bambara/”acha” with 15% Carrot; BAC20=Roasted bambara/”acha” with 20% Carrot;

56

4.11 Effect of Extrusion on the Vitamin Content of the Snacks

Effect of extrusion on the ß-carotene and C contents is presented in Table 15. There

was a significant (p<0.05) increase in vitamin A(as ß-carotene) with increase in carrot

inclusion in the blends.

Table 15: Effect of Processing on Vitamins A(as ß-c arotene) and C Contents of

the Extruded Snacks

Sample Code

Vitamin A (as ß-carotene) (µg/100g retinol)

Vitamin C (mg/100g)

BEFORE EXTRUSION BAB 53.08a±0.99 6.21a±0.25 BAC5 180.08b±0.85 7.60b±0.73 BAC10 278.46c±0.28 8.11b±1.01 BAC15 417.79d±0.74 8.67b±0.98 BAC20 548.52e±0.92 8.96b±0.65

AFTER EXTRUSION BAB 56.62a±1.10 2.54c±0.25 BAC5 181.33b±0.67 2.51c±0.44 BAC10 289.96c±0.54 3.92d±2.10 BAC15 419.00d±1.30 3.70d±0.86 BAC20 550.13e±0.22 4.05d±1.09 CEP 46.7 ND *RDA 700 45 *% RDA 25-80 6-9 ND –Not Detected. Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05). (*Source: US Committee on Dietary Allowances (1980)). RDA= Recommended Dietary Allowances

BAB=Roasted bambara/”acha” blend without carrot (control); BAC5=Roasted bambara/”Acha” blend with 5% Carrot; BAC10=Roasted BGN/”Acha” with 10% Carrot; BAC15=Roasted bambara/”Acha” with 15% Carrot; BAC20=Roasted bambara/”Acha” with 20% Carrot; CEP= Commercial extruded product

The formulation with 20% carrot (BAC20) had the highest level of ß-carotene (548.32

µg/100g retinol), while the BAB had 53.08µg/100g retinol content. The blends with 5%

carrot (BAC5) had 181.33µg/100g retinol, 10% (BAC10) had 278.46 µg/100g retinol

and 15% inclusion (BAC15) had 417.79µg/100g retinol. The ß-carotene content of the

CEP was found to be 46.7µg/100g, which was comparable to bambara-acha blend

without carrot (BAB) but much lower than the extruded samples with carrot that were in

the range of 180.08-550.13µg/100g. However, there was no significant difference

(p>0.05) in the ß-carotene content of these blends on extrusion, though there was a

slight increase in vitamin A after extrusion. Extrusion did not overtly affect the ß-

carotene level of the products. Rowe et al. (2009) reported similar observation. The ß-

carotene content of the extruded snacks in this study will would meet about 25 – 80% of

57

the RDA for ß-carotene in children, which would make it an ideal snack for vitamin A

Deficiency (VAD) feeding program. The observed stability of ß-carotene in this work

agrees with the report of Atwood et al. (1995) who noted that vitamin A exhibits good

stability during cooking. Vitamin C on the other hand is sensitive to heat and oxidation.

There was a significant reduction in the vitamin C content of the blends after extrusion.

In this study, over 50% of the vitamin C content was lost after extrusion. Vitamin C was

reduced from 6.21mg/100g in the control to 2.54mg/100g after extrusion. Sample BAC5

was reduced from 7.60mg/100g to 2.51mg/g, BAC10 from 8.11 to 3.92mg/100, BAC15

from 8.67 to 3.70 and BAC20 from 8.96 to 4.05mg/100g after extrusion. Vitamin C is

known to be extremely heat labile in a neutral pH and liquid matrix. Cooking losses

depend on the degree of heat, surface area exposed to water and oxygen, pH,

presence of transition metals and other factors that enhance oxidation (Anderson and

Hedlund, 1990; Eitenmiller and Landen, 1999). Anderson and Hedlund (1990) equally

reported that vitamin C decreased in wheat flour when extruded at fairly low (10%)

moisture. Gregory (1996) noted that it was not unusual for vitamin C cooking losses to

reach 100%. Ranum and Chome (1997) reported vitamin C levels of below detectable

limits in five of nine cooked Corn Soy Blend (CSB) samples collected in the field.

The vitamin B1 content of the blends ranged from 0.54 to 0.61mg/100g, B2 (0.39-

0.44mg/100g), vitamin B3 content ranged from 2.34 to 2.59mg/100g, while B6 was in the

range of 0.56 to 0.68mg/100g. The bambara /”acha” blends without carrot had slightly

higher content of all the B vitamins (B1,B2,B3 and B6) analysed. This could be probably

due to the higher content of these vitamins in bambara groundnut and acha compared

to carrot. However, extrusion cooking brought about significant reduction in these

vitamins, with vitamin B1 being the most affected, followed by vitamin B6. Extrusion

brought about between 40-50% reductions in vitamin B1, while about 25-30% reduction

was recorded in B6. Vitamins B2 and B3 recorded about 15-24% reductions in their

contents. Athar et al. (2006) reported a decrease in the levels of B group of vitamins

after extrusion of corn-pea blends, with thiamine and pyridoxine being the most affected

with over 50% reduction in thiamine content. Camire et al. (1990) and Killeit (1994)

obtained similar results. They reported that thiamine and pyridoxine were the most

thermolabile on extrusion and that the levels decreased linearly with temperature that

riboflavin was less sensitive to heat but more sensitive to shear. Compared to the

(commercial extruded product) (CEP) the extruded snacks had relatively lower

58

quantities of vitamins B1,B2,B3 and B6 probably because the market brand had been

fortified with these vitamins to meet specific needs as mandated by the Standard

Organization Nigeria (SON). The extruded snacks in this study were not fortified with

vitamin B complex.

Table 16: Effect of Extrusion on the Vitamin B Cont ent of Bambara-“Acha” Extruded Snacks Containing Grad ed Levels of Carrots (mg/100g) Samples B1

(Thiamin) B2

(Riboflavin) B3

(Niacin) B6

(Pyridoxine) Before Extrusion

BAB 0.61a±0.79 0.44a±0.38 2.59a±0.28 0.68a±0.65 BAC5 0.60a±0.41 0.42a±0.71 2.48a±0.03 0.64a±0.24 BAC10 0.55b±0.08 0.40a±0.55 2.53a±0.36 0.60b±0.46 BAC15 0.58ab±0.15 0.42a±0.86 2.45a±0.11 0.56b±0.18 BAC20 0.54b±0.50 0.39b±0.42 2.34a±0.70 0.61b±0.02

After Extrusion BAB 0.37c±0.32 0.35bc±0.11 2.04b±0.79 0.58b±0.04 BAC5 0.35cd±0.71 0.34c±0.85 2.00b±0.19 0.56b±0.01 BAC10 0.35cd±0.18 0.53bc±0.08 1.96c±0.82 0.50c±0.45 BAC15 0.36c±0.24 0.32c±0.40 1.92b±0.64 0.44c±0.21 BAC20 0.32d±0.53 0.33c±0.25 1.90b±0.05 0.46bc±0.33 CEP 0.99 1.08 12.36 1.41 *RDA 1.20 1.40 16.00 1.60

*% RDA 26-31 23-25 12-13 28-33

Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05). *Source: US Committee on Dietary Allowances (1980). RDA= Recommended Dietary Allowance BAB=Roasted bambara/Acha blend without carrot (control); BAC5=Roasted bambara/Acha blend with 5% Carrot; BAC10=Roasted BGN/Acha with 10% Carrot; BAC15=Roasted bambara/Acha with 15% Carrot; BAC20=Roasted bambara/Acha with 20% Carrot; CEP= Commercial extruded product

4.12 Effect of Storage on the Texture of the Extrud ates

The effect of storage period on the texture (crunchiness) of three extruded snack

samples (BAB, BAC5 and BAC10), which were selected for storage studies based on

their nutrient composition and sensory acceptability is presented in Table 17.

Table 17: Effect of Storage Period on the Texture ( Crunchiness) of Extruded Snacks

Sample Code

Storage Period (Months)

O 2 4 6

Compression force (N)

Energy (N/m)

Compression force (N)

Energy (N/m)

Compression force (N)

Energy (N/m)

Compression force (N)

Energy (N/m)

BAB 230.17a±0.25 0.36±0.81 214.48a±0.11 0.32±0.05 189.45b±1.23 0.28±0.09 176.21b±1.39 0.25±0.91

BAC5 218.09a±1.22 0.33±0.25 206.32a±0.81 0.29±0.64 190.82b±1.11 0.28±0.65 178.34b±0.43 0.24±0.50

BAC10 228.32a±0.88 0.34±0.211 210.05ab±0.99 0.31±0.02 198.11b±0.77 0.29±0.31 180.77c±1.07 0.26±0.22

Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05).BAB=Roasted bambara/”acha” blend without carrot (control); BAC5=Roasted bambara/”acha” blend with 5% Carrot; BAC10=Roasted BGN/”Acha” with 10% Carrot.

59

There were significant differences (p<0105) in the compression force (force required to

compress or break the extruded snacks) of the extruded snacks with increase in

storage period. The compression force decreased (p<0.05) after 4 months, which could

be attributed to softening due to slight increase in moisture content of the samples with

storage.

4.13 Effect of Storage on the Sensory Qualities of the Snacks

The sensory quality scores of the extruded snacks stored over a 6-month period under

ambient conditions (28±2°C) are presented in Table 18. There was no significant

difference (p>0.05) in colour/ appearance, taste, mouthfeel, flavour, crunchiness and

acceptability over the 6 months period of storage. The result however, revealed gradual

decrease in the mean scores of the attributes, though the changes were not significant

(p>0.05). The assessors were not able to detect any significant difference (p>0.05) in

crunchiness within the storage period, inspite of the slight increase in their moisture

content that ranged from 5.17 to 5.50% after 6 months. After 6 months of storage,

samples BAB and BAC5 had high sensory ratings for most of the quality attributes and

compared well with freshly prepared samples.

Table 18: Effect of Storage (6 months at 28±2°C) on the Sensory Qualities of

Extruded Snacks Samples Colour Taste Mouthfeel Flavour/

Aroma Crunchiness Overall

Acceptability 0 Month

BAB 7.5a±0.130 7.4a±0.85 7.0a±0.98 7.1a±1.06 7.6a±1.04 7.4a±1.27 BAC5 7.3a±0.71 7.5a±1.34 7.1a±0.39 7.3a±0.73 7.4a±0.85 7.29±0.99 BAC10 7.4a±0.99 7.19b±0.55 6.5a±0.54 7.0a±0.84 7.0a±0.76 7.0ab±1.26

2 Months BAB 7.4a±0.09 7.5a±0.45 7.1a±1.25 7.0a±0.85 7.5a±0.77 7.0a±0.99 BAC5 7.5a±0.22 7.2a±0.38 7.0a±0.49 6.8a±0.66 7.5a±0.52 7.0a±0.11 BAC10 7.2a±0.36 7.0a±0.69 6.9a±0.88 6.9a±0.21 7.0a±0.13 6.8a±0.41

4 Months BAB 7.0a±2.00 7.1a±0.99 6.8a±0.01 6.9a±0.94 7.0a±0.85 6.8a±0.74 BAC5 7.1a±0.45 7.0a±0.12 6.7a±0.36 7.0a±0.86 6.9a±92 6.4a±0.59 BAC10 7.0a±1.20 6.7a±1.09 6.3a±.81 6.6a±1.11 6.9a±0.91 6.5a±1.02

6 Months BAB 6.9a±1.27 7.0a±0.98 6.7a±0.46 6.2a±0.69 6.8a±0.76 6.7a±0.91 BAC5 7.1a±0.93 6.8a±0.56 6.5a±0.52 6.5a±1.20 6.9a±0.34 6.8a±0.86 BAC10 7.0a±1.01 6.7a±0.88 6.5a±1.35 6.7a±0.23 6.8a±0.45 6.6a±0.11 Column means±SD with different superscripts are significantly different at 5% probability level (p< 0.05). BAB=Roasted bambara/Acha blend without carrot (control); BAC5=Roasted bambara/Acha blend with 5% Carrot; BAC10=Roasted BGN/Acha with 10% Carrot.

Fig. 4 shows the effects of storage period under ambient conditions (28±2°C) and carrot

inclusion on the moisture content of the packaged extruded snacks. There was

60

significant difference (p<0.05) in the moisture content of the samples with increase in

storage period. The moisture content of sample BAB increased from 4.05% at O month

to 5.26% after 6 months storage, recording about 29.9% increase in moisture. The

moisture content of sample BAC5 increased from 4.25 to 5.17% with 22.20% moisture

absorption, while BAC10 increased from 4.31 to 5.50% recording 27% moisture

absorption. Shaw et al. (1994) also recorded increase in moisture content for extruded

snacks containing mechanically separated beef and potato flour. Samples stored for 6

months had significantly (p<0.05) higher moisture content than the freshly prepared

samples. The decrease in breaking strength or compression force could be attributed to

the effect of moisture absorbed by the samples during storage.

Fig.4: Effect of storage under ambient conditions (2 8±2°C) on the moisture content of packaged extruded snacks

0

1

2

3

4

5

6

0 2 4 6

Storage period, months

Moi

stur

e co

nten

t, %

BAB-Roasted BGN/"acha" withoutcarrot"

BAC5- Roasted BGN/"acha" with 5%carrot

BAC10- Roasted BGN/"acha with10% carrot

4.14 Effect of Storage on the Vitamin Content of th e Snacks

Table 19 shows the effect of storage period on ß-carotene content of the extruded

snacks. There was a gradual decrease or reduction in the ß-carotene content of the

extruded snacks during the storage period. At the 4th month of storage, the ß-carotene

content of all the samples was significantly (P<0.05) lower than the ß-carotene content

of the fresh samples. After 6 months storage, the ß-carotene content of extruded

bambara/”acha” without carrot was 19.0% lower than the fresh sample, the 5% (BAC5)

containing carrot samples was 23.8% lower while the 10 (BAC10) carrot containing

61

sample was 26.6% lower than the fresh sample. The decrease in ß-carotene content

during storage could be due to long exposure of the product to light, and also probably

due to the antioxidant properties/activities of ß-carotene in the product during storage

that may have reduced/affected ß-carotene composition of the product. Light has been

reported (Krause and Hunschar, 1972) to affect stability of ß-carotene.

Table 19: Effects of Storage (6 months) at 28± oC on the Vitamin A(as ß-carotene )

Content of Extruded Snacks

Sample Codes

O 2 4 6 % Loss (After

6 months) BAB 56.62a±1.10 54.01b±0.75 50.62c±1.84 45.89d±0.32 19.0 BAC5 180.78d±0.85 176.41c±0.25 169.95b±1.81 141.76a±0.99 21.6 BAC10 289.96d±0.54 283.20c±2.36 270.11b±1.55 212.43a±0.87 26.7

Values are mean±SD of triplicate determination. Column means with different superscripts are significantly different at 5% probability level (p< 0.05). BAB=Roasted bambara/”acha” blend without carrot (control); BAC5=Roasted bambara/”acha” blend with 5% Carrot; BAC10=Roasted bambara/”acha” with 10% Carrot.

62

CHAPTER FIVE

5.0 CONCLUSIONS AND RECOMMENDATI ONS

5.1 CONCLUSIONS

The work revealed that bambara groundnut, hungry rice and carrot, are rich in different

nutrients, which when combined/blended in the right proportions to make composite

flour, would produce nutrient dense flour and consequently food products rich in

protein, Vitamin A and other vitamins and minerals. The products would help to

alleviate the twin problems of protein energy malnutrition and micro-nutrient malnutrition

(hidden hunger) in Nigeria.

From the study it was established that:

1. Blending of bambara groundnut, hungry rice and carrot produced composite flour

rich in protein (12-15%), Vitamin A (181.33-548 µg/100g retinol) and Iron (6.90-

7.81mg/100g), which could be used for different food applications;

2. The composite flour produced very acceptable extruded snacks with high protein

(13-15%), vitamin A (180-550.13mg/100g retinol) and minerals (Iron and Zinc) that

could be exploited for school feeding programme;

3. Extrusion cooking of the blends brought about significant (p<0.05) increase in

protein digestibility and improvement on other nutrients such as minerals;

4. Extrusion cooking did not have significant (p>0.05) effect on the vitamin A and

mineral content of the extruded snacks. However, the treatment brought about

significant (p<0.05) reduction in phytate and tannin contents of the extruded snacks,

and totally destroyed trypsin inhibitor and haemagglutnin activities;

5. Extrusion cooking also brought about significant (p<0.05) reduction in vitamin C and

other heat labile B vitamins like thiamin and riboflavin;

6. Storage for 6 months at room temperature did not have adverse effect on vitamin A

and moisture contents, as well as on the sensory attributes (taste,

colour/appearance, flavour, crunchiness and overall quality).

5.2 RECOMMENDATIONS

Dietary diversification and blending of agricultural produce rich in different nutrients

should be encouraged in food product formulations and development, because it

reduces nutritional imbalance associated with diet from single produce.

The use of extrusion cooking should be encouraged and adopted in the production of

cereal-legume based snacks, because it improves protein quality and digestibility,

drastically reduces anti-nutritional factors and enhance product acceptability.

63

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Appendix 1

PHOSPHORUS STANDARD CURVE

y = 0.0186x

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 10 20 30 40 50 60 70 80 90

CONC(mg/100ml)

AB

S

75

Appendix 2

IRON STANDARD CURVE

y = 0.1306x

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10 12 14

CONC(µg/ml)

AB

S

76

Appendix 3

Mag nes ium s tandard c urve

y = 0.475x

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 1 2 3 4 5

C onc entration (µg /ml)

Ab

so

rba

nc

e

77

Appendix 4

PHYTATE STANDARD CURVE

y = 0.1283x

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10 12 14

CONC(µg/ml)

AB

S

78

Appendix 5

79

Appendix 6

Appendix 7

VITAMIN B1 STANDARD CURVE

y = 0.5604x

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

CONC(mg/100ml)

AB

S

80

Appendix 7

VITAMIN B2 STANDARD CURVE

y = 3.7041x

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.05 0.1 0.15 0.2 0.25CONC(mg/100ml)

AB

S

81

Appendix 8

Vitamin C standard curve

y = 18.585x

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 0.02 0.04 0.06 0.08 0.1 0.12

Concentration(mg/ml)

Abs

orba

nce

82

Appendix 9

VITAMIN B6 STANDARD CURVE

y = 0.0526x

0

0.1

0.2

0.3

0.4

0.5

0.6

0 2 4 6 8 10 12

CONC(mg/100ml)

AB

S