PRODUCTION and EVALUATION of BREAKFAST CEREALS from BLENDS of AFRICAN YAM BEAN (Sphenostylis Stenocarpa), MAIZE (Zea Mays) and DEFATTED COCONUT (Cocos Nucifera)

Home , Breakfast cereal, History of breakfast

PRODUCTION AND EVALUATION OF BREAKFAST CEREALS FROM BLENDS OF AFRICAN YAM BEAN (Sphenostylis stenocarpa), MAIZE (Zea mays) AND DEFATTED COCONUT (Cocos nucifera).

USMAN, GRACE OJALI

PG/M.Sc./09/50997

DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY, UNIVERSITY OF NIGERIA, NSUKKA.

NOVEMBER, 2012 i

TITLE PAGE

PRODUCTION AND EVALUATION OF BREAKFAST CEREALS FROM BLENDS OF AFRICAN YAM BEAN (Sphenostylis stenocarpa), MAIZE (Zea mays) AND DEFATTED COCONUT (Cocos nucifera).

A DISSERTATION SUBMITTED TO THE DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY, FACULTY OF AGRICULTURE, UNIVERSITY OF NIGERIA, NSUKKA, IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF M.Sc. IN FOOD SCIENCE AND TECHNOLOGY.

USMAN, GRACE OJALI

PG/M.Sc./09/50997

DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY, UNIVERSITY OF NIGERIA, NSUKKA.

NOVEMBER, 2012 ii

CERTIFICATION

USMAN, GRACE OJALI, a Post-graduate student in the Department of Food Science and

Technology, Faculty of Agriculture, University of Nigeria, Nsukka, with Registration

Number: PG/M.Sc./09/50997 has satisfactorily completed the requirements for award of the degree of Master of Science 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 G.I OKAFOR MR C.S. BHANDARY (SUPERVISOR) (HEAD OF DEPARTMENT)

------Date Date

iii

DEDICATION

This work is dedicated to the Holy Spirit, my source of inspiration and my family, for helping me in ways I can never quantify.

ACKNOWLEDGEMENTS

The successful completion of this research was made possible through the efforts and commitment of so many to whom I owe my appreciation. My foremost thanks go to the

Almighty God, who makes all things possible to them that believe in Him.

My sincere thanks goes to my Supervisor, Dr G.I. Okafor, whose advice, patience, dedication and relentless efforts led to the successful completion of this work. I am also grateful to, Mr.

C.S. Bhandary and the entire staff of the Department: Prof P. O. Ngoddy, Prof. T. M.

Okonkwo, Prof. (Mrs.) N. J. Enwere, Dr (Mrs.) J.C. Ani, Dr. P.O. Uvere, Dr. J. I. Eze, Dr.

(Mrs.) I. Nwaoha and Mrs. Omah, for imparting the knowledge and skills that equipped me throughout the period of this study and made this work a reality.

I owe my parents, Prof. and Mrs. S.S. Usman a lot of appreciation for their patience, encouragement, love and support, which motivated me at every stage of this work. I fondly appreciate my siblings, Adaji, Chide, Ugbede and Baby Praise for always being there for me.

I also extend my sincere appreciation to my brethren of the Graduate Students' Fellowship,

University of Nigeria, Nsukka for always making me feel at home.

Lastly, my profound gratitude goes to all my friends; Mary, Lucy, Toyin, Barrister, fellow professional colleagues and all those whose names are not mentioned. I love you all.

ABSTRACT Six samples were generated by mixing the flours (AYB+ maize composite) with graded levels of defatted coconut flour (100:0, 90:10, 80:20, 70:30, 60:40, 50:50), sugar, salt, sorghum malt extract and water. Breakfast cereals were produced by roasting (280°C) -a dry heat treatment process to gelatinize and semi-dextrinize the starch in order to generate dry ready to eat products from blends of African yam bean (Sphenostylis stenocarpa), maize (Zea mays) and defatted coconut (Cocos nucifera) cake. They were subjected to proximate, functional, sensory, minerals, vitamins, anti-nutrients, amino acids and microbial analyses. The products obtained were also served dry (without added water), with cold water, cold milk and warm milk to 15 panelists along with Weetabix (commercial control) to evaluate for appearance, consistency, flavour, taste, aftertaste, mouth feel, and overall acceptability using a 9 point Hedonic scale (1=dislike extremely, 9=like extremely). The results revealed the following ranges: proximate parameters (%): moisture (3.38-4.20), protein (15.68-18.26), fat (1.84-2.02), crude fiber (6.70-9.08), ash (5.29-7.36), carbohydrates (60.96-64.53), and energy (327.54-347.72Kcal). Functional properties were: pH (4.70- 6.56), bulk density (0.29- 0.71g/ml), water absorption capacity (68.31- 76.39%), oil absorption capacity (0.87- 1.32%), foam capacity (2.48- 3.49%), viscosity (19.73-31.08%), invitro-protein digestibility (66.30- 82.2%), and gelation capacity (75.32- 89.66%). Mineral analysis showed the following ranges (mg/100g): calcium (169-213), magnesium (290-430), potassium (88-191), manganese (5.92-7.99), iron (9.81-14.1), copper (0.58- 0.86), sodium (7.62- 9.97), zinc (2.11- 3.35). Vitamins analysis also revealed the following ranges (mg/100g): B1 (0.09-0.31), B2, (0.32-0.43), B6 (0.13- 0.26), B12 (0.74-1.01) and C (1.70- 2.65). Results for the anti-nutrients showed the following ranges (mg/100g): phytates, (0.38-1.25), oxalate (0.076-0.302), hemagluttinins, (0.10- 0.29) and tannins (0.00064-0.0016). Amino acids detected ranged as follows (mg/100g): phenylalanine (190-320), valine (160-240), threonine (560-810), tryptophan (380-520), isoleucine (110-220), methionine (10-100), histidine (160-240), arginine (180-510), lysine(90-250), leucine (590-810), cysteine (210-340), alanine (110-220), glycine (460-750), serine(80-120), aspartic acid (10-40), glutamic acid (10-40), asparagine (190-520), glutamine (100-300) and proline (30-50). Microbial analysis revealed the following ranges: bacteria count, 0.5x10 -1.51x102 Cfu/g, mold count, 0.0x10- 0.6x10 Cfu/g, while coliform was not detected. The sensory results revealed that the samples obtained were acceptable to the panelists, and there were no significant differences (p>0.05) between the control (Weetabix) and the samples in terms of overall acceptability when served with cold water, while significant (p<0.05) differences existed when served dry, with cold milk and hot milk. vi

TABLE OF CONTENTS

Page

Title page ------i Certification ------ii Dedication ------iii Acknowledgments ------iv Abstract ------v Table of Contents ------vi List of Tables ------ix List of Figures ------x Appendices ------xi

1.0 CHAPTER ONE: INTRODUCTION - - - - 1 1.1 Statement of Research Problem - - - - - 3 1.2 Significance of the study ------4 1.3 Objective of the Study ------4

2.0 CHAPTER TWO: LITERATURE REVIEW - - - 5 2.1 Breakfast and its importance - - - - - 5 2.1.1 Constituents of a Healthy Breakfast - - - - - 7 2.1.2 History of Breakfast Cereals ------7 2.1.3 Classification of Breakfast Cereals - - - - - 8 2.2 Cereals ------10 2.2.1 Maize Production and Utilization - - - - - 11 2.2.2 Varieties of Maize ------12 2.2.3 Nutritional Value of Maize ------12 2.3 Legumes ------13 2.3.1 World Production of Legumes - - - - - 13 2.3.2 Nutritional Relevance of Legumes - - - - - 14 2.3.3 Anti-nutritional Factors in Legumes - - - - - 14 2.4 Underutilized Legumes ------16 2.5 African Yam Beans (AYB) ------17 2.5.1 Nutrient Composition of African Yam Beans - - - 17 2.5.2 Potentials of African Yam Beans - - - - - 18 2.5.3 Factors Limiting the Use of African Yam Beans - - - 18 2.6 Coconut ------19 2.6.1 Origin and Morphology of Coconut - - - - - 19 2.6.2 Natural habitat of Coconut ------19 2.6.3 Nutritional Value of Coconut - - - - - 19 2.6.4 Coconut in Traditional and Modern Medicine - - - 21 2.6.5 Coconut as a Source of Dietary Fiber in Foods - - - 22 2.7 Production and Utilization of Sorghum - - - - 22 2.7.1 The use of Sorghum for the production of malt extract - - 23

3.0 CHAPTER THREE: MATERIALS AND METHODS - - 24 3.1 Material Procurement ------24 3.1.1 Sample Preparation ------24 3.1.2 Production of Maize Flour ------24 vii

3.1.3 Production of African Yam Beans Flour - - - - 26 3.1.4 Production of defatted Coconut flour - - - - 28 3.1.5 Production of Sorghum Malt Extract - - - - 30 3.2 Products Formulation ------32 3.3 Analysis of Samples ------35 3.3.1 Proximate Composition ------35 3.3.1.1 Determination of Moisture Content - - - - - 35 3.3.1.2 Determination of Crude Fat Content - - - - - 35 3.3.1.3 Determination of Protein Content - - - - - 36 3.3.1.4 Determination of total Ash Content - - - - - 36 3.3.1.5 Determination of Crude Fiber Content - - - 37 3.3.1.6 Determination of Carbohydrate - - - - - 37 3.3.1.7 Determination of Energy Value - - - - - 37 3.4 Functional Properties Determination - - - - 37 3.4.1 Determination of pH ------37 3.4.2 Determination of Bulk Density - - - - - 38 3.4.3 Determination of Water/ Fat Absorption Capacity - - - 38 3.4.4 Determination of Foam Capacity - - - - - 38 3.4.5 Determination of Viscosity ------38 3.4.6 Determination of In-vitro Protein Digestibility - - - 39 3.4.7 Determination of Gelation Capacity - - - - - 39 3.5 Sensory Evaluation ------39 3.6 Determination of Anti-nutritional Factors - - - - 40 3.6.1 Determination of Phytate or Phytic Acid - - - - 40 3.6.2 Determination of Tannin ------40 3.6.3 Determination of Oxalate ------41 3.6.4 Determination of Hemagluttinin - - - - - 41 3.7 Determination of Mineral content - - - - - 42 3.8 Determination of Vitamin content - - - - - 42 3.8.1 Determination of Vitamin B1 - - - - - 42 3.8.2 Determination of Vitamin B2 - - - - - 43 3.8.3 Determination of Vitamin B6 - - - - - 44 3.8.4 Determination of Vitamin B12 - - - - - 45 3.8.5 Determination of Vitamin C - - - - - 45 3.9 Determination of Essential and Non-essential Amino Acids - 46 3.10 Microbiological Examination - - - - - 46

4.0 CHAPTER FOUR: RESULTS AND DISCUSSION - - 47 4.1 Proximate Composition ------47 4.1.1 Moisture ------47 4.1.2 Protein ------49 4.1.3 Fat ------49 4.1.4 Ash ------49 4.1.5 Crude Fiber ------49 4.1.6 Carbohydrate ------50 4.1.7 Energy ------50 4.2 Functional Properties ------52 4.2.1 pH ------52 4.2.2 Bulk Density ------52 4.2.3 Water Absorption Capacity ------52 viii

4.2.4 Oil Absorption Capacity ------53 4.2.5 Foam Capacity ------53 4.2.6 Viscosity ------53 4.2.7 In-Vitro Protein Digestibility ------53 4.2.8 Gelatin Capacity ------54 4.3 Sensory Evaluation ------57 4.3.1 Attribute Perception of Samples Served Dry - - - - 57 4.3.2 Attribute Perception of Samples Served With Cold Water - - 59 4.3.3 Attribute Perception of Samples Served With Cold Milk - - 61 4.3.4 Attribute Perception of Samples Served With Hot Milk - - 63 4.3.5 Effect of Serving Style on Sensory Attributes of the Samples - 65 4.4 Mineral Composition of the Breakfast cereals - - - 70 4.4.1 Calcium ------70 4.4.2 Magnesium ------70 4.4.3 Potassium ------71 4.4.4 Manganese ------71 4.4.5 Iron ------71 4.4.6 Copper ------71 4.4.7 Sodium ------72 4.4.8 Zinc ------72 4.5 Vitamin Composition o f the Breakfast cereals - - - 74 4.5.1 Vitamin B1 ------74 4.5.2 Vitamin B2 ------74 4.5.3 Vitamin B6 ------74 4.5.4 Vitamin B12 ------75 4.5.5 Vitamin C ------75 4.6 Anti-Nutritional Factors ------77 4.6.1 Phytate/Phytic Acid ------77 4.6.2 Oxalate ------77 4.6.3 Hemagluttinin ------77 4.6.3 Tannin ------78 4.7 Amino Acid Profile ------80 4.8 Microbial Examination ------82

5.0 CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS 5.1 Conclusion ------84 5.2 Recommendations ------84

REFERENCES ------86

LIST OF TABLES Page

1 Average Contribution of Cereals and Cereal products to nutrient intake in the U.K. ------6

2 Proximate Composition of the Cereals grown in Nigeria - - - 11 3 Gross Chemical composition of different types of Maize - - - 13 4 Proximate composition of lesser known Legumes - - - 16 5 Coconut Dietary value per 100g edible portion - - - - 21 6 Composite flour formulations for Breakfast cereals made from blends of AYB + Maize: defatted coconut flour - - - - 33

7 Ingredients combination of Breakfast cereals made from blends of AYB + Maize: defatted coconut flour - - - - - 33

8 Proximate composition of Breakfast cereals made from blends of AYB + Maize: defatted coconut flour - - - - - 48

9 Functional properties of Breakfast cereals made from blends of AYB + Maize: defatted coconut flour - - - - - 55

10 Mean sensory scores for samples served dry - - - - 58 11 Mean sensory scores for samples served with cold water - - 60 12 Mean sensory scores for samples served with cold milk - - - 62 13 Mean sensory scores for samples served with hot milk - - - 64 14 Mineral content of Breakfast cereals made from blends of AYB + Maize: defatted coconut flour - - - - - 73

15 Vitamin content of Breakfast cereals made from blends of AYB + Maize: defatted coconut flour - - - - - 76

16 Anti-nutritional content of Breakfast cereals made from blends of AYB + Maize: defatted coconut flour - - - - - 79

LIST OF FIGURES Page 1 Taxonomy of the Graminae family - - - - - 10

2 Modified flow diagram for the production of Maize flour - - 25

3 Flow diagram for the production of African Yam Bean flour - - 27

4 Modified flow diagram for the production of defatted coconut flour - 29

5 Modified flow diagram for the production of malt extract - - 31

6 Flow diagram for the production of breakfast cereals from blends of AYB+Maize: Defatted coconut flour - - - - - 34

7 Energy values of breakfast cereals from blends of AYB + Maize: defatted Coconut Flour ------51

8 In-Vitro Protein Digestibility of breakfast cereals from blends of AYB+Maize: defatted Coconut flour - - - - - 56

9 Effect of the serving style on the colour perception of breakfast cereals from blends of AYB + Maize: defatted Coconut flour - - - 66

10 Effect of the serving style on the consistency perception of breakfast cereals from blends of AYB + Maize: defatted Coconut flour - - - 66

11 Effect of the serving style on the flavour perception of breakfast cereals from blends of AYB + Maize: defatted Coconut flour - - - 67

12 Effect of the serving style on the taste perception of breakfast cereals from blends of AYB + Maize: defatted Coconut flour - - - 67

13 Effect of the serving style on the aftertaste perception of breakfast cereals from blends of AYB + Maize: defatted Coconut flour - - - 68

14 Effect of the serving style on the mouthfeel perception of breakfast cereals from blends of AYB + Maize: defatted Coconut flour - - - 68

15 Effect of the serving style on the overall acceptability perception of breakfast cereals from blends of AYB + Maize: defatted Coconut flour - - 69

16 Amino acid profile of breakfast cereals from blends of AYB + Maize: defatted Coconut flour ------81

17 Microbial content of freshly prepared breakfast cereals from blends of AYB + Maize: defatted Coconut flour - - - - - 83 xi

APPENDICES Page

I Sensory Evaluation score sheet - - - - - 94

II Amino acid profile for formulated breakfast cereals - - 95

III Raw values for the Microbial profile of Breakfast Cereals made from blends of AYB+Maize: Defatted Coconut flour - - - 96

IV ANOVA Table for Anti-Nutrients of formulated Breakfast Cereals 97

V ANOVA Table for Sensory Data of formulated Breakfast Cereals served Raw ------98

VI ANOVA Table for Sensory Data of formulated Breakfast Cereals served with cold water ------99

VII ANOVA Table for Sensory Data of formulated Breakfast Cereals served with cold milk ------100

VIII ANOVA Table for Sensory Data of formulated Breakfast Cereals Served with Hot Milk ------101

IX ANOVA Table for Functional Properties Analysis - - 102

X ANOVA Table For Proximate Composition Analysis - - 103

XI ANOVA Table for Vitamin Analysis - - - - 104

XII RDA of Vitamins for Children and Adults (mg/kg of body weight) 105

XIII RDA for Mineral requirements for Children and Adults - - 106

XIV RDA of Essential Amino Acids for Children and Adults - - 107

CHAPTER ONE

1.0 INTRODUCTION

The word “breakfast” is a compound of "break" and "fast" which literally means “breaking the fast” from the last meal or snack from the previous day. Breakfast is the nutritional foundation or the first meal of the day (Kowtaluk, 2001). Nutritional experts have referred to breakfast as the most important meal of the day, citing studies that found people who skip breakfast to be disproportionately likely to have problems with concentration, metabolism, and weight (Mayo Clinic, 2009). Breakfast meals vary widely in different cultures around the world. It often includes a carbohydrate source such as cereals, fruit and or vegetable, protein, sometimes dairy, and beverage.

In developing countries, particularly sub-Saharan Africa, breakfast meals for both adults and infants are based on local staple diet made from cereals, legumes, and cassava and potatoes tubers. However, the most widely eaten breakfast foods are cereals (Kent, 1983).

Breakfast cereals are legally defined as foods obtained by swelling, grinding, rolling or flaking of any cereal (Sharma and Caralli, 2004). They can be categorized into traditional (hot) cereals that require further cooking or heating before consumption and ready-to-eat (cold) cereals that can be consumed from the box or with the addition of milk (Fast 1990; Tribelhorn, 1991). Ready to eat breakfast cereals are increasingly gaining acceptance in most developing countries, and gradually displacing most traditional diets that serve as breakfast due to convenience, nutritional values, improved income, and status symbol and job demands especially among urban dwellers. According to Jones (2003), instantized and ready-to-eat (RTE) cereals facilitate independence because of their ease of preparation which means that children and adolescents can be responsible for their own breakfast or snacks. Such foods may need to be reconstituted, pre-heated in a vessel or allowed to thaw if frozen before consumption, or they may be eaten directly without further treatment (Okaka, 2005). The common cereal products in Nigeria include NASCO Cornflakes, Good morning corn flakes, Kellogg‟s cornflakes, NABISCO flakes, Weetabix, Quaker Oats, Rice crisps, among others. A study has clearly shown that 42% of 10-year-olds and 35% of young adults consumed cereal at non-breakfast occasions (Haines et al., 1996). This may be consumed dry as snack food, with or without cold or hot milk, based on their location, availability of resources and habits. 2

In recent times food product developers have incorporated legumes into traditional cereal formulations as nutrient diversification strategy as well as efforts to reduce the incidence of malnutrition among vulnerable groups. Results from previous studies (Onweluzo and Nnamuchi, 2009), indicated that most cereals are limited in some essential amino acids especially threonine and tryptophan. Though cereals are rich in lysine (especially the yellow maize), they cannot effectively provide the nutrients required by the body, especially in the morning when the supply of nutrients from the previous day is exhausted. Cereals can however, be supplemented with most oil seeds and legumes which are rich in essential amino acids particularly the sulphur-containing ones (Kanu et al., 2007). Thus a combination of such food stuffs will improve the nutritional value of the resulting blend compared to the individual components alone. Animal products such as meat, eggs, milk, and cheese are known to contain the essential amino acids that could complement this deficiency in cereal foods. However, consumption of proteins from plant sources (Legumes) is encouraged (Ofuya and Akhidue, 2005), since combination of legumes and grains provide biologically high quality and cheaper protein that contains all essential amino acids in proper proportion and their amino acids complement each other (Okaka, 2005).

Legumes or pulses are edible fruits or seeds of pod bearing plants (Sivasanka, 2005). Their seeds are put to a myriad of uses, both nutritional and industrial, and in some parts of the developing world they are the principal source of protein for humans (Trevor et al., 2005). Legumes have high protein content, in the range of 20-40%; about twice that of cereals and several times that in root tubers (Sivasanka, 2005). The common legumes in Nigeria include, Cowpea (Vigna unguiculata), Soybeans (Glycine max), Pigeon pea (Cajanus cajan), Groundnuts (Arachis hypogea), African yam bean (Sphenostylis stenocarpa), etc. (Okaka, 2005).

A variety of legumes, including African yam bean (Sphenostylis stenocarpa) are under exploited or underutilized (Ebiokpo et al., 1998). African yam bean is the most economically important among the seven species of Sphenostylis (Potter, 1992). It is a lesser- known legume of the tropical and sub-tropical areas of the world which has attracted research attention in recent times (Azeke et al., 2005). It is a climbing legume with exceptional ability for adaptation to low lands and takes about five to seven months to grow and produce mature seeds (Apata and Ologhoba, 1990). AYB seeds can be brown, white, speckled or marbled with a hilum having a dark-brown border. The seeds form a valuable and prominent source of 3 plant proteins in the diet of Nigerians and are cultivated as a pulse for human consumption. The Ibo people of the south eastern Nigeria call it “Okpodudu, Ijiriji, Azama” and the seeds may be boiled and eaten with local seasoning, starchy roots, tubers and fruit or converted to paste for the production of a type of “moi-moi”. The seeds can also be roasted and eaten with palm kernels (Enwere, 1998). AYB, a non-conventional pulse has been brought into focus by some previous workers as it is known to have a nutritive and culinary value (Agunbiade and Ojezele, 2010).

Nutritionists recommend 20-35 grams of dietary fiber a day which could be obtained from sources of dietary fiber such as whole grains, legumes, and nuts. Coconut is an excellent source of dietary fiber, which has been made available as a dietary supplement (Bruce Fife, 2010). Coconut dietary fiber is made from finely ground, dried, and defatted coconut and has higher fiber content than many other fiber supplements.

Formulating a breakfast cereal with blends of these raw materials highlighted above could bring about diversification in the utilization of indigenous underutilized food crops for national sustenance.

1.1 STATEMENT OF RESEARCH PROBLEM African yam bean has been recognized to have vast genetic and economic potentials, especially in reducing malnutrition among Africans; however the crop has not received adequate research attention, thereby limiting its contribution to food security and preventing potential food crisis. Increasing the use of underutilized crops is one of the better ways to reduce nutritional, environmental and financial vulnerability in times of change (Jaenicke and Pasiecznik, 2009).

Over time, some conditions have negatively influenced the productivity and acceptability of African yam bean among cultivators, consumers, and research scientists. These include, characteristic hardness of the seed coat (Oshodi et al., 1995) which increases the cost and time of cooking, presence of anti-nutritional factors (ANF) or secondary metabolites (Machuka and Okeola, 2000) and the tendency to cause flatulence in humans (Rockland and Nishi, 1979). Therefore, it is of interest to process African yam bean seeds into acceptable, ready-to-eat and safe products together with other locally available materials including maize and defatted coconut flour. 4

1.2 SIGNIFICANCE OF THE STUDY African yam bean has been reported to have equal or higher lysine content than that of Soybean while most of other essential amino acids correspond to the WHO/FAO recommendation (Yetunde et al., 2009). In addition to this, it is reported to be important in the management of chronic diabetes, hypertension and cardiovascular diseases because of its low glycemic index and high dietary fiber content (Enwere, 1998). This research, therefore, has the potential to address the twin problems of energy malnutrition as well as food security. It will stimulate establishment of food industries for the production of breakfast cereals and create other marketing and employment opportunities.

1.3 OBJECTIVE OF THE STUDY The general objective of this study is to produce and evaluate breakfast cereals from blends of African yam bean (Sphenostylis stenocarpa), maize (Zea mays) and defatted coconut (Cocos nucifera).

Specific objectives:

1. To produce flours from African yam bean, maize and defatted coconut. 2. To produce breakfast cereals from blends of African yam bean, maize and defatted coconut. 3. To evaluate the chemical, functional, sensory, and microbial as well as the anti- nutrients, minerals, vitamins and amino acid profile of the products obtained.

CHAPTER TWO 2.0. LITERATURE REVIEW

2.1.0 BREAKFAST AND ITS IMPORTANCE

Breakfast is the most important meal of the day and breakfast cereals are the most nutrient- dense, tasty, convenient and typically lowest calorie breakfast options (Hochberg-Garrett, 2008). The importance of breakfast in assuring adequate nutrient intakes has been documented in numerous studies both in the United States and elsewhere (Yan Want, et al., 1992). Should breakfast be omitted, food consumption during the rest of the day may not provide sufficient nutrients to meet the recommended dietary allowances (RDAs) for vitamins and minerals (Preziosi et al., 1999).

Previous studies define a time range for breakfast consumption, such as 5 AM to 9 AM. For example, a study with children in developed countries used 5 AM to 10 AM on weekdays and 5 AM to 11 AM on weekends. Although the choices for breakfast foods are endless, a few foods remain the most popular items for this meal. It was found that in some developed and developing countries three most popular breakfast items for adults were coffee, milk, and breads and the three most popular breakfast items for children were milk, cereal, and juice. At this time, 75% of participants reported eating breakfast at home, 22% ate away from home, and 3% ate at both places (Hochberg-Garrett, 2008).

Eating breakfast has been shown to be beneficial for both body and mind in several ways. Those who eat a cereal-based breakfast (including pre-sweetened cereals), have a lower body mass index (BMI) than those who skip breakfast or choose an alternative breakfast option (Hunty and Ashwell, 2006). The average contribution of cereals and cereal products to nutrient intake is shown in Table 1. It has been proven in many studies that those who eat breakfast have a lower BMI than those who skip breakfast. Below are some interesting facts relating to breakfast cereals which were cited by Nicklas (2004) and Hunty and Ashwell (2006). i. Eating breakfast contributes to cognitive performance and improves concentration ii. Breakfast cereals supply one quarter of essential micronutrients to children‟s diets. iii. Breakfast cereals are an essential source of iron for teenagers iv. Breakfast cereals provide an important source of folic acid as well as increasing levels of Vitamin D, B-vitamins and minerals including zinc and iron. 6

v. Breakfast cereals are also an important source of calcium both through the product itself and the addition of milk to the cereal. vi. Recent studies by the Irish Universities nutrition Alliance has also shown that breakfast cereals provide teenagers with up to 11% of their daily fiber requirements.

Breakfast is the most commonly skipped meal among children. Many reasons were given for adults and children to skip breakfast, such as poverty and parental influences (Hochberg- Garrett, 2008). It has been observed that children who do not have their breakfast before leaving for school have problems, like headache, sleepiness, stomach pain, muscle fatigue, etc. (Kartha, 2010). Indecisiveness, anger, anxiety, irritability, unhappiness, nervousness, lethargy, hostility, etc. are some other problems that can be seen in students who skip their breakfast. Such physical and psychological problems have the ability to hinder the learning process; students who have their breakfast regularly score better in their tests than those who avoid eating breakfast (Jegtvig, 2008). A small study in adults also found that a high-fiber carbohydrate-rich breakfast was associated with the highest post-breakfast alertness rating and the greatest alertness between breakfast and lunch. A larger study found an association between breakfast cereal consumption and subjective reports of health, with those adults who ate breakfast cereal every day reporting better mental and physical health, compared to those who consumed it less frequently (McKevith, 2004).

Table 1. Average Contribution of Cereals and Cereal Products to Nutrient Intake in the (UK %).

Nutrient Boys Girls Adults

Energy 35 33 31 Protein 27 26 23 Carbohydrate 45 42 45 Fat 22 21 19 NSP 40 37 42 Thiamin 43 38 34 Riboflavin 34 31 24 Niacin 38 34 27 Folate 44 37 33 Vitamin B6 30 26 21 Vitamin D 37 35 21 Iron 55 51 44 Calcium 27 27 30 Sodium 40 38 35 Potassium 15 14 12 Source: McKevith, 200 7

2.1.1 CONSTITUENTS OF A HEALTHY BREAKFAST Though carbohydrates which provide energy to the body is one of the most important parts of breakfast, it is necessary to make sure that the breakfast is not wholly a carbohydrate meal. A complete breakfast should include all the necessary nutrients, including proteins, calcium, vitamin B6, vitamin A, zinc and iron (Jegtvig, 2008). Also, it would contain low level of sodium, salt and sugar. A basic breakfast should be nothing less than cereal, milk and fruits.

2.1.2 HISTORY OF BREAKFAST CEREALS The history of breakfast cereals has been summarized by Carson (1957) and Wikipedia (2009). Breakfast cereals have their beginnings in the vegetarian movement in the last quarter of the nineteenth century, which influenced members of the Seventh-day Adventist Church in the United States. The main Western breakfast at that time was a cooked breakfast of eggs, bacon, sausage, and beef. The first packaged breakfast cereal, granular (named after granules) was invented in the United States in 1863 by James Caleb Jackson, operator of the Jackson Sanitarium in Dansville, New York and a staunch vegetarian. The cereal never became popular; it was far too inconvenient, as the heavy bran nuggets needed soaking overnight before they were tender enough to eat. Ferdinand Schumacher, president of the American Cereal Company, created the first commercially successful cereal made from oats; manufacturing took place in Akron, Ohio.

In 1877, John Harvey Kellogg, invented a biscuit made of ground-up wheat, oat, and cornmeal for his patients suffering from bowel problems. The product was initially also named "Granula", but changed to "Granola" after a lawsuit. His most famous contribution, however, was an accident. After leaving a batch of boiled wheat soaking overnight and rolling it out, Kellogg had created wheat flakes. His brother Will Keith Kellogg later invented corn flakes from a similar method, bought out his brother's share in their business, and went on to found the Kellogg Company in 1906. In the 1930s, the first puffed cereal, Kix, went into the market. Beginning after World War II, the big breakfast cereal companies – now including General Mills, who entered the market in 1924 with Wheaties – increasingly started to target children. The flour was refined to remove fiber, which at the time was considered to make digestion and absorption of nutrients difficult, and sugar was added to improve the flavor for children. The new breakfast cereals began to look starkly different from their ancestors. Today, breakfast has gained much ground in the food industry. 8

2.1.3 CLASSIFICATION OF BREAKFAST CEREALS Breakfast cereals fall into the class of convenient foods. These can be regarded as foods which have been fully or partially prepared, in which significant preparatory input, culinary skills and energy have been transferred from the home maker‟s kitchen to the food processor‟s factory. Such foods may need to be reconstituted, pre-heated in a vessel or allowed to thaw if frozen before consumption, or they may be eaten directly without further treatment (Okaka, 2005).

The classification of the breakfast cereals are based on the amount of heat required for its preparation. According to Kent (1975), breakfast cereals can be classified according to: a. The amount of domestic cooking required b. The form of product or dish c. The cereal used as raw material.

Those breakfast cereals that require cooking are of four types. The endosperm of the grains may, sometimes, simply be broken or pressed, with or without toasting, to yield such uncooked cereals. They include, i. Entire grain such as rice ii. Flaked such as rolled oats iii. Coarsely ground such as hominy grits iv. Finely ground such as cream of wheat

The breakfast cereals which need no cooking are called ready-to-eat cereals. For these the endosperm of the cereal grain may be broken or ground into a mash, and then converted into flakes by squeezing shapes; or the endosperm may be kept intact as kernels to be puffed as in the case of puffed rice. In all cases, the flaked, formed or puffed cereals are oven cooked and dried to obtain a toasted flavour and to obtain the crisp, brittle textures desired (Potter and Hotchkiss, 2006). These cereals are sold in many forms e.g.: a. Flaked cereals from corn, wheat and rice b. Puffed cereals from rice and wheat c. Shredded cereals from wheat d. Granular cereals from most cereals. 9

The grains can be puffed, flaked, extruded, pelleted, shredded or produced in a granular form and have sugar, honey or vitamin added. The ready-to-eat bran is combined with raisins or prunes. Also, classification could be according to the manner in which the meals are served whether they are in the ready-to-serve form (for example cornflakes) or whether they require some cooking before being served (e.g. porridge). The ready-to-serve breakfast cereals may be classified into hot cereal, whole-grain cereals, bran cereals, sugary cereals and organic cereals based on the manufacturing methods (Wikipedia, 2009).

In the market today whether cereal is hot or cold, conventional or organic, the possibilities for good nutrition are seemingly endless. Cereals are presented in various types which appeal to the eye as well as the appetite of the consumer. Below are the different types of cereals found in both national and international market as compiled by Kinsey (2009).

Hot Cereal: Options such as oatmeal, Cream of Wheat and Malt-O-Meal are healthy hot breakfast that fall into this category. They come in wholesome, unsweetened versions as well as in sugary, processed versions. By buying unsweetened, whole-grain hot breakfast cereals, one can add healthier natural sweeteners such as honey and fruit.

Whole-Grain Cereal: Whole-grain cereals, such as Cheerios, Kashi and Shredded Wheat fall into this category. The whole grains have very little or no added sugars. Researchers at Columbia University Medical Center have found that oat-based whole grain cereals can help reduce cholesterol and aid in heart health. Other whole grains, such as whole wheat, can help an individual feel full and satisfied as the day begins.

Bran Cereal: cereals, such as Raisin Bran, Fiber One and Bran Flakes are in this category and are high-fiber breakfast cereals. Fiber can give the feeling of fullness and aid in digestion and regularity.

Sugary Cereal: Sugary cereals are often placed at a child's eye level in the grocery store. These cereals are often highly processed and have loads of added sugar and preservatives. Cereals such as Reese's Puffs, Fruit Loops and Lucky Charms can be eaten as an occasional fun treat, but if an adult or child eats them on a daily basis, they might notice that the huge sugar rush affects their mood and energy level.

Organic Cereal: Nature's Path, EnviroKidz and Cascadian Farm are popular organic cereal brands. These brands produce cereals similar to most popular conventional cereals, and they do it using ingredients free of pesticides and fertilizers. Organic foods also cannot be 10 genetically engineered. Most cereals use natural sweeteners that are not overly-processed as well as lots of whole grains.

2.2.0 CEREALS Cereals are fruits of cultivated grasses belonging to the monocotyledonous family Graminae. The principal cereal crops of the world are wheat, barley, oats, rice, rye, maize, sorghum and millets but the chief cereals in the developing countries in West Africa are maize, rice, sorghum and millets. The taxonomy of the Graminae family is shown in Figure 1. Wheat is the principal protein source of the world, followed by maize, rice, oats, soybeans, etc. (National Research Council, 1988). The most commonly grown ones in Nigeria are sorghum, millet, maize, rice and wheat. These five crops occupy an estimated measure of over 16 million hectares of farmland (Okoh, 1998). The anatomical structure of all cereals grains is basically similar differing from one another in details only. Of the important grain cereals, maize, sorghum, naked grain millets and rice (tropical cereals) and wheat (temperate cereal), have a fruit coat (pericarp) and seed. The seed comprise the seed coat, germ and endoplasm (Okaka, 2005).

Figure 1: Taxonomy of the Graminae Family Source: Shewry et al., (1992)

In the tropics, cereals are the staple foods of the people providing about 75% of their total caloric intake and 67% of their total protein intake (Adedeye and Adewoke, 1992). Table 2 shows the proximate composition of the main cereals grown in Nigeria. In the Northern part of Nigeria, cereals are the main sources of protein and energy. These grains are consumed in many forms as pastes, roasts, porridges, gruels and pottages or other preparation of the seed which when milled flour, bran oil, starch, breakfast or dinner cakes as well as breakfast cereals are obtained. Cereals therefore offer a better source of protein other than the root crops in the diet of Nigerians; whole protein intake from animal source is low (Ihekoronye and Ngoddy, 1985).

Table 2: Proximate Composition of the main Cereals grown in Nigeria (% dry matter basis)

Cereal Protein Fat Carbohydrate Crude Fiber Mineral Salt

Maize 10.50 5.40 68.00 2.40 1.60 Sorghum 9.28 2.27 85.20 2.01 1.24 Millet 13.69 5.39 77.26 1.80 1.96 Rice 7.07 2.25 89.89 0.23 0.56 Wheat 11.63 2.33 81.91 2.97 1.16 Acha 6.96 2.10 87.48 1.02 2.44

Source: Mbaeyi, 2005

2.2.1 MAIZE PRODUCTION AND UTILIZATION The origin of maize is considered to be America, particularly southern Mexico. USA is one of the major corn producing countries in the world with a production of more than 50% of the world crop. This share however has decreased from about 40% to about 25% because of the development of high yielding strains of hybrid maize. Maize is extensively cultivated in India, both in the plains and in the hill regions (Shakuntala and Shadaksharswamy, 2001). Maize (Zea mays L.) is the most important cereal in the world after wheat and rice with regard to cultivation (Osagie and Eka, 1998). In sub-Saharan Africa maize is a staple food for an estimated 50% population. It is an important source of carbohydrate, protein, iron, vitamin B, and minerals. More than 40 different ways of consuming maize had been recorded in many countries in Africa (Nago et al., 1990). Africans consume maize as a starchy base in a wide variety of porridges, pastes, grits, and beer. Green maize (fresh on the cob) is eaten parched, baked, roasted or boiled with or without salt and plays an important role in filling the hunger gap after the dry season (Nicklin, 2004). Every part of the maize plant has 12 economic value: the grain, leaves, stalk, tassel, and cob can all be used to produce a large variety of food and non-food products (Wikipedia, 2009).

2.2.2 VARIETIES OF MAIZE The principal maize varieties are flint corn, dent corn, sweet corn, pop corn, flour corn and waxy corn (Shakuntala and Shadaksharswamy, 2001). This classification is based on the nature and distribution of starch in the endosperm. Flint corn has very hard kernels. The texture is due to a rather thick layer of starch and proteins just under the bran layer. Flints mature early and are grown mostly in India. Dent corn has hard starch at the sides, while the major part of the endosperm contains soft starch. At maturity, a typical dent-lie depression appears at the crown. They are grown mostly in the USA. Sweet corn has a large proportion of carbohydrates of the kernel as dextrin and sugar in the unripe kernels are tender. When matured and dried, the kernels are hard and have a wrinkled surface. The major part of the endosperm of the pop corn comprises of starch on all sides, with a very small core of soft starch. The flour corn grains are large and soft and the endosperm is very friable. These characteristics permit easy grinding of the corn into flour (Wikipedia, 2009). Also, the waxy corn contains a high proportion of amylopectin and is of industrial importance.

2.2.3 NUTRITIONAL VALUE OF MAIZE

Maize or corn grains consist of the outer hull or bran which contains a lot of fiber, embryo (germ) rich in oil and the endosperm rich in starch. Whole maize contains about 11% protein, 4% fat, 3% fibre, 65% of starch and other carbohydrates and 1.5% of minerals (Sivasankar, 2005; Ihekoronye and Ngoddy, 1985). Maize is deficient in the mineral niacin. Maize is milled to separate the outer layer and the germ from the endosperm. The germ is recovered to obtain germ oil, a valuable product used as salad oil. Maize bran and the oil cakes are used as animal feed. The starchy endosperm separated during milling is used to make flour and other traditional products. Larger grits obtained by screening are used for making corn flakes and porridge. Corn starch is hydrolyzed to give glucose and high fructose corn syrup. The chemical composition of different varieties of maize is illustrated in Table 3.

Table 3: Gross Chemical Composition of Different types of Maize (%)

Maize type Moisture Ash Protein Crude Ether Carbohydrate fibre extract

Salpor 12.2 1.2 5.8 0.8 4.1 75.9

Crystalline 10.5 1.7 10.3 2.2 5.0 70.3

Floury 9.6 1.7 10.7 2.2 5.4 70.4

Starchy 11.2 2.9 9.1 1.8 2.2 72 8

Sweet 9 5 1 5 12.9 2.9 3.9 69.3

Pop 10.4 1.7 13.7 2.5 5.7 66.0

Black 12.3 1.2 5.2 1.0 4.4 75.9

Source: Cortez and Wild-Altamirano, 1972

2.3. LEGUMES

The term legume, is derived from the Latin word legumen (with the same meaning as the English term), which is in turn believed to come from the verb legere "to gather." English borrowed the term from the French "legume," which, however, has a wider meaning in the modern language and refers to any kind of vegetable; the English word legume being translated in French by the word legumineuse (Wikipedia, 2009).

2.3.1 WORLD PRODUCTION OF LEGUMES There are over 13,000 species of plants belonging to this family. Some are cultivated as crop plant whose seed are edible (Shakuntala and Shadaksharaswamy, 2001). Over the years wide varieties of legumes have been domesticated. In this process, ancient Indian and Chinese civilization seem to have played an important role in some legume species (Soybean, Bengal gram, etc.). The world‟s second largest producers of pulses is India coming next only to China, with the production of 14.2 million tones cultivated in an area of 24.4 million hectares with an average yield of 6.02 quintals per hectare (Sivasanka, 2005).

The legumes used for food are divided into two groups; pulses and oil seeds. Pulses are dried edible seeds of cultivated legumes such as peas, beans and lentils. The second group, the oil seeds, consists of those legumes used primarily for their oil content which may be extracted 14 by pressing or by solvent extraction, the residue being high oil cake. These include the groundnuts and the soybeans (Ihekoronye and Ngoddy, 1985).

2.3.2 NUTRITIONAL RELEVANCE OF LEGUMES Legumes are critical to the balance of nature; for many are able to fix atmospheric Nitrogen to ammonia with the aid of nodular bacteria. A leguminous crop can add up to 500g of Nitrogen to the soil per hectare annually (Okaka, 2005). The potentials of legumes as a protein source, especially in regions where meat production is inadequate or is inexistent have long been recognized (Aykroyd and Doughty, 1982). The nutritional value of legumes is related to their high protein content (12-25%). Legumes contain relatively low quantities of the essential amino acid methionine. To compensate, some vegetarian cultures serve legumes along with grains, which are low in the essential amino acid lysine, which legumes contain. Thus a combination of legumes with grains can provide all necessary amino acids for vegetarians. Common examples of such combinations are „dal with rice‟ by Indians, and beans with corn tortillas, tofu with rice, and peanut butter with wheat bread (as sandwiches) in several other cultures, including Americans (Vogel, 2003).

The enrichment of cereal based foods with legumes and oilseeds has received considerable attention. In Nigeria, the high cost of commercial industrially produced high protein energy rich breakfast products make them out of reach to low income earners, consequently people in this wage category who constitute an appreciable percentage of the population depend for their breakfast on left over super or at best on sole cereal porridge that is of low nutritional value. There is therefore the need to develop affordable low cost high protein energy breakfast product whose production would not require high technology (Onweluzo and Nnamuchi, 2009).

2.3.3 ANTI-NUTRITIONAL FACTORS IN LEGUMES Notwithstanding the agronomic and nutritional advantages of legumes as cheap protein sources for many, especially low income persons, legumes have been reported to contain several anti-nutritional factors which include hemaglutinins, neurotoxic factors such as β- aminopropionitril which cause lethrism. Other anti-nutrients in legumes are hemolytic-fibrile factor, as contained in faba beans, which causes favism, goitrogenic factors and trypsin inhibitors (Okaka, 2005; Liener, 1983 and Osho, 1989). The anti-nutritional factors are segregated into two major groups based on their responses to heat treatment. One group, 15 which includes protease inhibitors, lectins (hemagglutinins), goitrogens and anti-vitamin factors are heat labile, while the other group which include saponins, eastrogens, lysino- alanines, allergens, flatulence inducing factors and phytates are heat stable and need treatments other than heat or other treatments in combination with heat to reduce their negative effects on man and animals (Liener, 1980 ; Okaka, 2005). Some of these anti- nutrients are explained below: PHYTATES: Phytic acid phosphorus constitutes the major portion of total phosphorus in several seeds and grains. It accounts for 50–80% of the total phosphorus in different cereals. It was reported by some authors (Schwenke et al., 1989) that phytic acid level has no or very little effect on binding to proteins. The investigation of the possibility of formation of ternary complexes raises difficulties. At alkaline pH values the Ca-phytate is insoluble and forms precipitate. At very high pH values the phytate is insoluble. From a nutrition point of view, many studies have concentrated on the metal ion chelating property of phytic acid, its binding of zinc and formation of less soluble complexes that reduce zinc availability (Carnovale et al., 1988). PROTEASE INHIBITORS: All legumes have been found to contain trypsin inhibitors to varying degrees, in addition to chymotrypsin inhibitors. Inhibition of trypsin and chymotrypsin leads to the hypertrophy of pancreas (Enwere, 1998). Conditions of heating- time and temperature, moisture content, and particle size- influence the rate and extent of trypsin inhibitor inactivation (Enwere, 1998). HEMAGGLUTININS: These are also referred to as lectins. Their occurrence is not limited to legumes alone as they are found in slime molds, fungi, lichens, other flowering plants and animals such as crustaceans, snails, fish, amphibian eggs and mammalian tissues (Enwere, 1998). Crude raw extract of hemagglutinin agglutinates the red blood cells of human beings and other animals if injected directly to the blood stream. Thus, it impairs the utilization of legumes such as beans, groundnuts, among others (Enwere, 1998).

The other set back that has limited the use of legumes in non-traditional food formulations is the objectionable flavour associated with the crops. This set back has been a primary focus of research in a bid to extend the use of some legumes. The most common off-flavour producing factors are the presence of glucosides-isoflavones, saponins, and sapogenols (Okaka, 2005).

2.4.0 UNDERUTILIZED LEGUMES

Lesser known and utilized legunes in Nigeria can be loosely divided into two classes - those which are prepared and eaten as other legumes (pigeon pea, bambara groundnuts, and African yam beans) and those which are not eaten as other legumes but may be used as thickeners, stabilizers or processed into condiments (akparata, achi, ofor, Ukpo) or fermented food products (African locust bean, castor oil seeds) (Enwere, 1998). The proximate composition of lesser known legumes is shown in Table 4.

Table 4: Proximate Composition of Some Lesser known Legumes (%)

Legume Moisture Crude Crude Ash Crude Total content protein fat fiber carbohydrates

Pigeon pea 67.40 7.0 0.60 1.3 3.50 20.20

(Unripe dried 10.10 19.2 1.50 3.8 8.10 65.40

African yam bean seed) 6.40 21.8 1.30 2.2 4.70 63.60

Bambara 9.70 16.0 5.90 2.9 ND 64.90 groundnut

Afzelia africana 5.28 27.04 31.71 3.22 ND 33.09

Deuterium 6.14 13.52 13.81 2.20 ND 64.26 microcarpiurn

Mucuna 5.84 20.41 -9.64 3.12 ND 61.10 flagellipes

Brachystegia 6.49 10.47 8.48 2.68 ND 71.94 eurycoma

Source: Mbaeyi, (2005)

Under-explored legumes are important in terms of food security, nutrition, and agricultural development, enhancement of economy and also as rotation crops. Thus, little known legumes can play an important role in agriculture as they are potent plants, which contribute to the world food production due to their adaptation to adverse environmental conditions and high resistance to diseases and pests (Sridhar and Seena, 2006). 17

2.5 AFRICAN YAM BEANS (AYB)

AYB belongs to the family Fabaceae, sub-family Papilionoideae, tribe Phaseoleae, sub-tribe Phaseolinae, and genus Sphenostylis (Allen and Allen, 1981). The crop has twining vigorous vines, which could be green or pigmented red. The vines twine clockwise around the stakes or climb other supports to a height of about 3m or more. The leaves are compound trifoliate. The large pink and purple flowers are admirable and attractive ornamentals, while the pods are usually linear, housing about 20 seeds. These vary in size, shape, colour, colour pattern, etc. The origins of AYB as indicated by GRIN (2009) includes the following countries within the tropical regions of Africa: Chad and Ethiopia (Northeast tropical Africa); Kenya, Tanzania and Uganda (East tropical Africa); Burundi, Central African Republic and Democratic Republic of Congo (West-Central tropical Africa); Côte d‟Ivoire, Ghana, Guinea, Mali, Niger, Nigeria, and Togo (West tropical Africa); Angola, Malawi, Zambia, and Zimbabwe (South tropical Africa). The centre of diversity of AYB is only within Africa. Nigeria is very significant for AYB production where extensive cultivation had been reported in the eastern, western, and southern areas of Nigeria. In different yield trials in Nigeria (IITA, Ibadan and Nsukka), the most productive accession in each case gave 1860 kg and 2000 kg of seeds/hectare (Adewale and Dominique, 2009).

2.4.1 NUTRIENT COMPOSITION OF AFRICAN YAM BEAN

The African yam bean is grown for both its edible seeds and its tubers. It is a vigorous vine, which twines and climbs to heights of about 3 m and requires staking. It flowers profusely in 100 to 150 days, producing brightly-coloured flowers, which may be pink, purple or greenish white. The slightly woody pods contain 20 to 30 seeds, are up to 30 cm long and mature within 170 days. The plant produces underground tubers that are used as food in some parts of Africa and serve as organs of perennation in the wild (Porter 1992). The chemical composition shows that it contains 21 - 29% protein, 5 - 6% crude fiber, 74.1% carbohydrate, 1.2% fat, 3.2% ash. (NAS, 1979). The proximate composition of the bean's hull shows a reasonably high crude protein (11.4%) but very low contents of crude fat (2.6%), phytic acid (82 mg/100 g) and phytin-phosphorus (23 mg/100 g). K and Ca are the major minerals present in yam bean hull. The hull, rich in cell wall polysaccharides, is composed of cellulose (35.4%); non-cellulose fractions made of pectin and hemicellulose put together (41.9%) and lignin(3.6%) (Agunbiade and Longe, 1998). Researchers (Uguru and Madukaife 2001) who did a nutritional evaluation of 44 genotypes of AYB reported that the crop is well balanced in essential amino acids and has higher amino acid content than pigeon pea, cowpea, and 18

Bambara groundnut.

2.4.2. POTENTIALS OF AFRICAN YAM BEANS

Food and Nutrition: The economic potentials of AYB are immense. Apart from the production of two major food substances, the value of the protein in both tubers and seeds is comparatively higher than what could be obtained from most tuberous and leguminous crops. The protein in the tuber of AYB is more than twice the protein in sweet potato (Ipomea batatas) or Irish potato (Solanum tuberosum) and higher than those in yam and cassava (Amoatey et al., 2000). Moreover, the amino acid values in AYB seeds are higher than those in pigeon pea, cowpea, and Bambara groundnut (Uguru and Madukaife, 2001). Protein content is up to 19% in the tuber and 29% in seed grain The content of crude protein in AYB seeds is lower than that in soybean, but the amino acid spectrum indicated that the level of most of the essential amino acids especially lysine, methionine, histidine, and iso-leucine in AYB compares favorably with whole hens‟ eggs and most of them meet the daily requirement of the Food and Agriculture Organization (FAO) and World Health Organization (WHO) (Ekpo, 2006). AYB is rich in minerals such as K, P, Mg, Ca, Fe, and Zn but low in Na and Cu (Nwokolo, 1987).

Insecticidal and Medicinal Usefulness: AYB as a crop is less susceptible to pests and diseases compared with most legumes; this quality may undoubtedly be due to the inherent lectin in the seed of the crop (Adewale and Domonique, 2009). Omitogu et al. (1999) advanced the prospect that the lectin in the seed of the crop is a promising source of a biologically potent insecticide against field and storage pests of legumes. Therefore, the inclusion of the lectin extract from AYB in the meal for three cowpea insect pests, namely, Maruca vitrata, Callosobruchus maculatus, and Clavigralla tomentosicollis gave a mortality rate greater than 80% after 10 days.

2.4.3. FACTORS LIMITING THE USE OF AFRICAN YAM BEANS

Over time, some conditions have negatively influenced the productivity and acceptability of this crop among cultivators, consumers, and research scientists. Notable among the list are, i) The characteristic hardness of the seed coat (Oshodi et al., 1995) which makes a high demand on the cost and time of cooking, ii) The agronomic demand for stakes, the long maturation period, and 19 iii) The presence of anti-nutritional factors (ANF) or secondary metabolites (Machuka and Okeola, 2000).

2.5 COCONUT 2.5.1 ORIGIN AND MORPHOLOGY

The English name coconut, first mentioned in English print in 1555, comes from the Spanish and Portuguese word coco, which means "monkey face." Spanish and Portuguese explorers found a resemblance to a monkey's face in the three round indented markings or "eyes" found at the base of the coconut (Filippone, 2007). The Coconut (Cocos nucifera), is an important member of the family Arecaceae (palm family). It is the only accepted species in the genius Cocos (Wikipedia, 2009) and is a large palm growing up to 30m tall, with pinnate leaves 4- 6m long and pinnae 60-90 cm long.

2.5.2 NATURAL HABITAT OF COCONUT

The Coconut palms are grown throughout the tropics (Ihekoronye and Ngoddy, 1985). They thrive on sandy soils and are highly tolerant of salinity. They prefer areas with abundant sunlight and regular rainfall (150 cm to 250 cm annually), which makes colonizing shorelines of the tropics relatively straightforward (Wikipedia, 2009). Coconuts also need high humidity (70–80%) for optimum growth, which is why they are rarely seen in areas with low humidity, like the Mediterranean, even where temperatures are high enough (regularly above 24°C or 75.2°F). Coconut trees are very hard to establish in dry climates, and cannot grow there without frequent irrigation; in drought conditions, the new leaves do not open well, and older leaves may become desiccated; fruit also tends to be shed (Wikipedia, 2009). Coconut palms are grown in more than 80 countries of the world, with a total production of 61 million tons per year (FAO, 2009).

2.5.3 NUTRITIONAL VALUE OF COCONUT

The coconut provides a nutritious source of meat, juice, milk, and oil that has fed and nourished populations around the world for generations. On many islands coconut is a staple in the diet and provides the majority of the food eaten. Nearly one third of the world's population depends on coconut to some degree for their food and their economy. Among these cultures the coconut has a long and respected history. Coconut is highly nutritious and rich in fiber, vitamins, and minerals. It is classified as a "functional food" because it provides many health benefits beyond its nutritional content. The coconut palm is so highly valued by them as both a source of food and medicine that it is called "The Tree of Life." Several food 20 uses or products exist for coconut. The primary product is copra, the white "meat" found adhering to the inner wall of the shell. It is dried to 2.5% moisture content, shredded, and used in cakes, candies, and other confections. Alternatively, coconut oil is expressed from copra, which is used in a wide variety of cooked foods and margarine. The raw copra can be grated and squeezed to obtain coconut "milk". Coconut water is obtained from immature coconuts, providing a welcome source of fresh, sterile water in hot, tropical environments. The sap from the cut end of an inflorescence produces up to a gallon per day of brown liquid, rich in sugars and vitamin C. It can be boiled down into a brown sugar called "jaggery", used as a sugar substitute in many areas. Left to ferment, the sap makes an alcoholic toddy, and later vinegar; "arrack" is made by distilling the toddy. Per capita consumption of coconut is 0.6 lbs/year. Coconut oil is probably consumed in greater quantities than confectionary coconut products, but coconut oil would be only a small percentage of the 47 pounds of vegetable oils consumed annually. Table 5 shows the dietary value of the edible portion of coconut. 21

Table 5: Coconut Dietary Value, per 100g edible portion

Dry coconut Coconut (copra) water Water (%) 3.3 95 Calories 556 19 Protein (%) 3.6 0.7 Fat (%) 39.1 0.2 Carbohydrates (%) 53.2 3.7 Crude Fiber (%) 4.1 1.1 % of US RDA* Vitamin A 0.8 0 Thiamin, B1 <1 0 Riboflavin, B2 <1 0 Niacin <1 0 Vitamin C 0-7 5.3 Calcium 5.4 3.0 Phosphorus 23.9 2.5 Iron 36 3.0 Sodium 0.4 2.4 Potassium 16.4 5.3 * Percent of recommended daily allowance set by FDA, assuming a 154 lb male adult, 2700 calories per day.

2.5.4 COCONUT IN TRADITIONAL AND MODERN MEDICINE In traditional medicine around the world coconut is used to treat a wide variety of health problems including the following: abscesses, asthma, baldness, bronchitis, bruises, burns, colds, constipation, cough, dropsy, dysentery, earache, fever, flu, gingivitis, gonorrhea, irregular or painful menstruation, jaundice, kidney stones, lice, malnutrition, nausea, rash, scabies, scurvy, skin infections, sore throat, swelling, syphilis, toothache, tuberculosis, tumors, typhoid, ulcers, upset stomach, weakness, and wounds (Bruce-Fife, 2010).

Modern medical science is now confirming the use of coconut in treating many of the above conditions. Published studies in medical journals show that coconut, in one form or another may provide a wide range of health benefits. Some of these are summarized below: It kills viruses that cause influenza, herpes, measles, hepatitis C, SARS, AIDS, and other illnesses. It also kills bacteria that cause ulcers, throat infections, urinary tract infections, gum disease and cavities, pneumonia, and gonorrhea, and other diseases. It kills fungi and yeasts that cause candidiasis, ringworm, athlete's foot, thrush, diaper rash, and other infections and expels or kills tapeworms, lice, giardia, and other parasites. It provides a 22 nutritional source of quick energy. It also boosts energy and endurance, enhancing physical and athletic performance (Bruce-Fife, 2010).

2.5.5. COCONUT AS A SOURCE OF DIETARY FIBER IN FOODS

Coconut dietary fiber is made from finely ground, dried, and defatted coconut meat. It has a mild great-tasting coconut flavor. Gunathilake et al. (2009) reported that coconut flour can provide not only value added income to the industry, but also a nutritious and healthy source of dietary fiber. Coconut flour may play a role in controlling cholesterol and sugar levels in blood and prevention of colon cancer. Studies revealed that consumption of high fiber coconut flour increases fecal bulk (Arancon, 1999).

Unlike many fiber sources, coconut dietary fiber does not contain phytic acid and, therefore, does not remove minerals from the body. Not only does coconut fiber not remove minerals, but it also increases mineral absorption. Coconut fiber slows down the rate of emptying food from the stomach. This allows food more time in the stomach to release minerals, leading to higher levels of minerals available for the body to absorb (Wasserman, 2010).

A tablespoon or two of coconut dietary fiber can be added to beverages, smoothies, baked goods, casseroles, soups, and hot cereal. This is a simple and easy way to add fiber into daily diet without making drastic changes in the way food is eaten. Another way to add coconut fiber into a diet is during baking. Up to 20% of the wheat in a recipe can be replaced with coconut fiber (Gunathilake et al., 2009). Coconut dietary fiber has all the benefits of other dietary fibers, it lowers risk of heart disease, helps prevent cancer, improves digestive function, helps regulate blood sugar, etc. (Bruce-Fife, 2010). It also has several advantages over most other forms of fiber including relieving symptoms associated with Crohn's disease, expel intestinal parasites, and improve mineral absorption (Guarner, 2005).

2.6.0 PRODUCTION AND UTILIZATION OF SORGHUM Sorghum (Sorghum bicolor L. Moench) is a warm season crop, intolerant of low temperatures but fairly resistant to serious pests and diseases. It is known by a variety of names (such as great millet and guinea corn in West Africa, kafir corn in South Africa, jowar in India and kaoliang in China) and is a staple food in many parts of Africa, Asia, and parts of the Middle East. Most of the sorghum produced in North and Central America, South 23

America and Oceania is used for animal feed (FAO, 1995). Sorghum (Sorghum bicolor L. (Moench) is a cultivated tropical cereal grass. It is generally, although not universally, considered to have first been domesticated in North Africa, possibly in the Nile or Ethiopian regions as recently as 1000 BC (Kimber, 2000). The cultivation of sorghum played a crucial role in the spread of the Bantu (black) group of people across sub-Saharan Africa (Taylor, 2004).

2.6.1 USE OF SORGHUM FOR THE PRODUCTION OF MALT EXTRACT The potential of sorghum as an important source of industrial brewing material has been long recognized. Indeed, during the World War II, sorghum was offered as a brewing adjunct because the conventional brewing material (barley) was scarce (Odibo et al., 2007). An important advantage of sorghum is that it can yield crop under harsh environmental conditions such as drought, where temperate cereals like barley fail to grow. An attempt to malt barley at a temperature higher than 18 °C showed that endosperm modification of barley was sub-optimal because enzyme development was inadequate (Odibo et al., 2007).

In southern Africa, malting sorghum for opaque beer brewing has developed into a large scale commercial industry with some 150,000 tonnes of sorghum being commercially malted annually. This figure includes a small amount of sorghum malted for the production of a sorghum malt breakfast cereal “Maltabela”. Sorghum is also malted commercially on a large scale in Nigeria for the production of lager beer and stout and for non-alcoholic malt-based beverages (Taylor, 2004).

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 MATERIAL PROCUREMENT Sound Maize grains (Zea mays L), African yam bean seeds (Sphenostylis stenocarpa), mature Coconut (Cocos nucifera L), salt, white Sorghum and sugar were purchased from Ogige market, Nsukka in Enugu state, Nigeria.

3.1.1. SAMPLE PREPARATION Maize grains and African yam bean seeds was properly cleaned and sorted to remove stones, dirt, chaff, weeviled seeds and other extraneous matters, before they were used for further processing.

3.1.2. PROCESSING OF MAIZE GRAINS INTO FLOUR The method used was a modification of the method described by Iheoronye and Ngoddy (1985) and Okaka (2005). 5kg of maize was cleaned and sorted after which it was milled into flour. The flow diagram for the production of whole maize flour is shown in Figure 2.

Maize Grains

Cleaning

Dry milling

WHOLE MAIZE FLOUR

Figure 2: Modified Flow diagram for the production whole maize flour (Source: Ihekoronye and Ngodddy, 1985).

3.1.3. PRODUCTION AFRICAN YAM BEAN FLOUR The procedure as described by Enwere (1998) was used. 5kg of cleaned/sorted brown African yam bean seeds were weighed and washed thoroughly with clean tap water after which they were soaked for 12 hours and boiled for 30 minutes. The beans were dried in a hot air oven (60oC for 10hours), dehulled and milled using an attrition mill. The flour obtained was sieved using 0.5mm mesh sieve and packaged in polyethylene bags for further analysis. The flow diagram for the production of raw fine African yam bean flour is shown in Figure 3.

African yam beans

Cleaning

Washing

Boiling

Drying

Dehulling

Milling

Sieving

Fine African yam bean flour

Figure 3: Flow diagram for the production of African yam bean flour (Source: Enwere, 1998)

3.1.4. PRODUCTION OF DEFATTED COCONUT FLOUR The procedure used was a modification of a method described by Sanful (2009). 3kg freshly dehusked Coconut was properly cleaned and cracked to expel the liquid content. The coconut flesh (meat) was removed from the shell with the aid of a sharp pointed knife. The brown colour of the skin was scraped off with a knife. The coconut flesh was grated using a manual grater, homogenized in boiling water (100oC) and poured into a muslin cloth and squeezed to obtain the defatted coconut paste that was further rinsed with hot water (>70oC) till the filtrate became colourless. The defatted coconut was then dried (60oC for 10hours) in the hot air oven, packaged in a polythene bag and sealed for further analysis. The flow diagram for the production of defatted coconut is shown in Figure 4.

Dehusked Coconut

Cracking

De-shelling

Grating

Homogenization

Sieving/pressing

Drying

Milling

Defatted Coconut flour

Figure 4: Modified Flow Diagram for the Production of Defatted Coconut Flour. (Source: Sanful, 2009)

3.1.5 PRODUCTION OF SORGHUM MALT EXTRACT The modification of the procedure described by Okafor and Aniche (1980) was used. Malting

5kg of white Sorghum grains were steeped in tap water for 18 h and germinated on floor for o three days at room temperature (28+ 20C). The green malt was then kilned at 55 C for 8 hours o and further at 65 C for 16 hours until the shoots and roots were friable and were separated from the grains.

Mashing: Three step decoction method was used to mash the sorghum malt during which 70% of the mash was maintained at 55oC for 30 minutes and at 65oC for 1 h and lastly at 70oC for 1 hour in a hot water bath. The conditioned mash was strained through a clean muslin cloth and the filtrate (malt extract) stored for use. The flow chart for the production of Sorghum malt is shown in Figure 5.

White Sorghum grains

Cleaning

Steeping

Malting

Drying

De-rooting

Milling

Mashing/heating

Cooling

Straining

Sorghum malt extract

Figure 5: Modified Flow Diagram for the production of Sorghum malt extract (Source: Okafor and Aniche 1980).

3.2 PRODUCTS FORMULATION Composite flour was formulated by mixing AYB and maize flour (60:40). Six samples of breakfast cereals were generated by mixing the composite flour (made of AYB: Maize flours) with graded levels of defatted coconut flour (100:0, 90:10, 80:20, 70:30, 60:40, 50:50), sugar, salt, sorghum malt extract and water, and roasted at 280°C with continuous stirring till dried products were obtained. A control sample was produced from 100% maize and African yam bean composite flour as shown in Table 6.

The ingredient combination of the breakfast cereals formulation is shown in Table 7 and Figure 6 shows the flow chart for the production of a roasted breakfast cereal.

Table 6: Composite Flour Formulations for Breakfast Cereals made from Blends of AYB+Maize: Defatted Coconut Flour. Sample Sample code Code Ratio Percentage (%) A AYB+M: DF 100:0 40% M, 60%AYB B AYB+M: DF 90:10 90%AYB+M, 10%DC C AYB+ M: DC 80:20 80% AYB+M, 20%DC D AYB+ M: DC 70:30 70% AYB+M, 30%DC E AYB+ M: DC 60:40 60%AYB+M, 40%DC F AYB+M: DC 50:50 50%AYB+M, 50%DC

M: Maize, AYB: African yam bean, DC: Defatted coconut flour

Table 7: Ingredients Combination for Breakfast Cereals made from Blends Of AYB+Maize: Defatted Coconut Flour per 100g SAMPLES Ingredient A B C D E F

M+AYB 84 74 64 54 44 34

DC - 10 20 30 40 50

Malt extract 10 10 10 10 10 10

Sugar 5 5 5 5 5 5

Salt 1 1 1 1 1 1

Legend: A= 100:0, B=90:10, C=80:20, D=70:30, E=60:40, F=50:50 AYB = African yam bean, M = Maize, DC = Defatted Coconut

Composite flour

Mixing with other ingredients

Addition of water

Roasting (285oC, 5mins)

Cooling

Packaging

Breakfast Cereal

Figure 6: Flow diagram for the Production of Breakfast Cereal from Blends of African Yam Bean + Maize: Defatted Coconut flour.

3.3 ANALYSES OF SAMPLES The following analyses were carried out on the six samples obtained. i. Proximate analyses. ii. Determination of the functional properties iii. Sensory Evaluation. iv. Determination of Anti-nutritional factors. v. Minerals determination. vi. Vitamins determination vii. Essential and non-essential amino acids determination viii. Microbiological examination

3.3.1 PROXIMATE COMPOSITION 3.3.1.1 DETERMINATION OF MOISTURE CONTENT The standard method of AOAC (2006) was used. Cleaned crucibles were dried in a hot air oven at 100oC for 1 hour to obtain a constant weight and then cooled in a dessicator. Two grams of each of the samples was then weighed into the different crucibles and dried at 100oC until a constant weight is obtained.

%moisture content = W2-W3 X 100 W2-W1

Where, W1 = Initial weight of the empty crucible W2 = weight of dish + sample before drying W3 = weight of dish + sample after drying.

3.3.1.2 DETERMINATION OF CRUDE FAT CONTENT Fat content was determined by the Soxhlet extraction method of AOAC (2006). A Soxhlet extractor with a reflux condenser and a 500ml round bottom flask was fixed. Two grams of the sample was then weighed into a labeled thimble. Petroleum ether (300ml) was filled into the round bottom flask and the extractor thimble was sealed with cotton wool. The Soxhlet apparatus was the allowed to reflux for about 6 hours after which the thimble was removed. Petroleum ether was collected from the flask after which it was dried at 105oC for 1hour in and oven cooled in a dessicator and weighed. This procedure was carried out for all the samples.

%fat = weight of fat X 100% weight of sample

Where, F = Percent fat content x1 = Initial weight of flask and sample x2 = Final weight of flask

3.3.1.3 DETERMINATION OF CRUDE PROTEIN This was determined using the micro-Kjeldahl method (AOAC, 2006). One gram weight of each flour sample was weighed into an l00ml Kjeldahl flask. 2.5 grams of anhydrous Na2S04,

0.5 gram of CUSO4 and 5ml of concentrated H2S04 was added and allowed to stand for 2-3 hours. The flask was then heated in a flame chamber, gently boiling initially for fumes to appear and heated more intensely until the solution is clear. After cooling, the content was transferred into an l00ml volumetric flask and made up to the mark with repeated washing using distilled water.

Distillation: A 5ml volume of each sample digest was mixed with 5ml of Boric acid indicator and 3 drops of methyl red in an l00ml conical flask and then steam distilled into conical flask using l00ml of 60% NaOH. Distillation was done for 5 minutes until colour changed from purple to green. 5ml distillate was collected and titrated against 0.01N HC1 to a purple colored endpoint. The percentage protein was calculated with this expression:

% Nitrogen = T x 14.01 x 0.01 x 20 x 100 1.0 x 100

Where T = Titre value 1 .0g = Weight of the sample 20 = Dilution factor (i.e. from 10015) 0.01 = Normality of HCl 14.01 = Atomic mass of nitrogen % Protein = %Nitrogen x 6.25 (where: 6.25 => Conversion factor of protein).

3.3.1.4 DETERMINATION OF TOTAL ASH Ash content was determined by AOAC (2006) procedure. Two grams of well blended samples was weighed into a shallow ashing dish (a crucible) that had been ignited, cooled in a dessicator and weighed soon after reaching room temperature. Both the crucibles and their content were transferred into a muffle furnace ignited at 550°C. Ashing was done for 8 hours; 37 crucible and the ashed sample were removed from the muffle furnace, moistened with a few drops of water to expose the un-ashed carbon, dried in the oven at 100°C for 4 hours and re- ashed at 550°C for another hour. These were removed from muffle furnace, cooled in a dessicator and weighed soon after reaching room temperature. Percentage ash was calculated using this expression:

% Ash = Weight of ash X 100% Weight of sample used

3.3.1.5 DETERMINATION OF CRUDE FIBER Crude fiber was determined by AOAC (2006) method. Two grams of the sample was weighed and put in a boiling 200ml of 1.25% H2SO4 and allowed to boil for 30minutes. The solution was then filtered through linen or muslin cloth fixed to a funnel. It was washed with boiling water until it is completely free from acid. The residue was returned into 200ml boiling NaOH and allowed to boil for 30 minutes. It was further washed with 1% HCl boiling water to free it from acid. The final residue was drained and transferred to a silica ash crucible dried in the oven to a constant weight and cooled. Percent crude fiber was calculated using the expression:

% Crude fiber = Loss in weight on ignition X 100 Weight of food sample

3.3.1.6 DETERMINATION OF CARBOHYDRATE CONTENT (BY DIFFERENCE) The total carbohydrate content was estimated as the difference between 100 and the total sum of moisture, fat, protein, crude fiber and ash as described by AOAC (2006).

3.3.1.7 DETERMINATION OF TOTAL ENERGY The total energy was determined by the method described by Kanu et al. (2009). The total energy or the caloric values was estimated by calculation using the water quantification factors of 4, 9 and 4kcaV100g respectively for protein, fat and carbohydrate.

3.4. FUNCTIONAL PROPERTIES DETERMINATION 3.4.1 pH DETERMINATION The pH of the food samples was measured with a Mettler Delta 350 pH meter using the method described by Onwuka (2005). The sample homogenates was prepared by blending 38 l0g sample in l00ml of deionized water. The mixture was filtered and the pH of the filtrate was measured. Triplicate readings were taken for each sample.

3.4.2 BULK DENSITY DETERMINATION Bulk density was determined for each of the formulated samples using the method described by Onwuka, (2005). Each sample was slowly filled into l0ml measuring cylinder. The bottom of the cylinder was gently tapped on a laboratory bench until there is no further diminution of the sample after filling to l0ml mark. Bulk density was estimated as mass per unit volume of the sample (g/ml). Triplicate measurements were taken.

3.4.3 DETERMINATION OF WATER AND OIL ABSORPTION CAPACITY (WAC/FAC)

The Water and Fat absorption capacities of the formulated samples were determined using the method described by Onwuka (2005). 1g of each of the samples was weighed into a conical graduated centrifuge tube, and then a warring whirl mixer was used to thoroughly mix the sample with 10ml of distilled water or oil for 30minutes. The mixture was allowed to stand for 30minutes at room temperature and then centrifuged at 5000xg for 30minutes. The volume of free water or oil (supernatant) was read directly from the graduated centrifuge tube. The absorption capacity was expressed as gram of oil or water absorbed (or retained) per gram of sample.

3.4.4 DETERMINATION OF FOAM CAPACITY The Foam capacity was determined using the method described by Onwuka (2005). Two grams of each of the formulated samples were blended with 100ml distilled water in a warring blender (the suspension was whipped at 1600rpm for 5minutes). The mixture was then poured into a 250ml cylinder and the volume after 30 seconds was recorded. The foam capacity was calculated using the formula; FC = Volume after whipping – Volume before whipping x 100 Volume before whipping

3.4.5 DETERMINATION OF VISCOSITY The viscosity of the samples was determined using the method described by Onwuka (2005) method. 10% of each formulated sample was suspended in distilled water and mechanically stirred for 2hours at room temperature. Oswald type viscometer was used to measure the viscosity of the mixture.

3.4.6 DETERMINATION OF IN-VITRO PROTEIN DIGESTIBILITY The invitro-protein digestibility of each sample was determined using the method described by Kanu et al. (2009). Five grams of each of the formulated samples was weighed into a 5ml centrifuge tube and to which 15ml of 0.1N HCl containing 1.5mg pepsin-pancreatin was added. The tube was incubated at 37oC for 3hours. The suspension was then neutralized with a phosphate buffer (pH 8.0) containing 0.005M sodium azide. 1ml of toluene was added to prevent microbial growth and the mixture was gently shaken and incubated for an additional 24hours at 37oC. After incubation, samples were treated with 10ml of 10% trichloroacetic acid (TCA) and centrifuged at 5000rpm for 20minutes at room temperature. The protein in the supernatant liquid was estimated using Kjedahl method. The percentage of protein digestibility was calculated using the formula;

Protein digestibility (%) = Protein in the supernatant x 100 Protein in the sample

3.4.7 DETERMINATION OF GELATION CAPACITY The gelation capacity was determined using the method described by Onwuka (2005). 2-20% W/V suspension of each of the samples was prepared in 5ml distilled water in test tubes. The sample test tubes were heated for 1hour in a boiling water bath which was followed by rapid cooling under running cold tap water. The test tubes were further cooled for 2hours at 4oC. The least gelation concentration was determined as that concentration at which the sample from the inverted test tube did not fall down or slip visually.

3.5.0 SENSORY EVALUATION The six formulated samples were served to 15 untrained panelists consisting of students of the University of Nigeria, about 10.00 am along with Weetabix (commercial control) using a 9 point Hedonic scale (1=dislike extremely, 9=like extremely). The samples were served raw/dry, with cold water, cold milk and warm milk and assessed for appearance, consistency, flavour, taste, aftertaste, mouth feel, and overall acceptability. The sensory scores obtained were further subjected to a one-way Analysis of Variance (ANOVA). The Least Significant Difference (LSD) test and Duncan Multiple Range Tests were used to determine significant differences between means and separate means respectively at p<0.05 levels using SPSS package version 17.0.

3.6.0 DETERMINATION OF ANTI-NUTRITIONAL FACTORS 3.6.1 DETERMINATION OF PHYTATE OR PHYTIC ACID The phytate determination was as described by Thompson and Erdman (1982). Two grams of each of the formulated samples was placed in a flask into which 100.0ml of 1.2 HCl and 10%

Na2S04 were added. The flask was stoppered and shaken for 2-hours on a mechanical shaker. The extract was vacuum filtered through No4 Whatman paper. 10.0ml of the filtrate was pipetted into a 50ml centrifuge tube. l0ml deionized water was added, followed by 12ml of

FeCl3 solution (2.0g FeCl3.6H2O) + 16.3ml conc. HCl per litre). The contents were stirred, heated for 75 minutes in boiling water and cooled, covered for one hour at room temperature. The tube was centrifuged at 1000Xg for 15 minutes. The supernatant was decanted and discarded and the pellet was thoroughly washed thrice with a solution of 0.6% HC1 and 2.5%

Na2S04. After each wash, the contents were centrifuged at 1000Xg for 10 minutes and the supernatant discarded. l0 ml concentrated HNO3 was added to the resulting pellet and the content transferred quantitatively to a 400ml beaker with several small portions of deionized water. 4 drops of concentrated H2SO4 was added and contents heated approximately 30 minutes in a hot plate until only the H2SO4 is left. Approximately 4 - 5ml of 30% H2O2 was added and the mixture returned to the hot plate at a low heat until bubbling ceases. The residue was dissolved in 15ml 3N HCl and heated for 10-15 mixtures. The resulting solution was made up of 100.0ml volume diluted 15 and then analyzed for iron using Franson et al., (1975) procedure.

3.6.2 DETERMINATION OF TANNIN The Folin-Denis colorimetric method as described by Kirk and Sawyer (1998) was used for the determination of tannin content in the samples as follows: 5g of the samples was dispersed in 50ml of distilled water and agitated. The mixture was allowed to stand for 30 minutes at room temperature and shaken every 5 minutes. After 30 minutes it was centrifuged and the extract obtained. The extract (2ml) was taken into a 50ml volumetric flask. Similarly, 2ml standard tannin solution (tannic acid) and 2ml of distilled water was put in separate 50ml volumetric flask to serve as standard and reagent (1.0ml of Folin-Denis) added to each of the flasks, followed by addition of 2.5ml of saturated sodium carbonate solution. The content of each flask was made up to 50ml with distilled water and allowed to incubate for 90 minutes at room temperature. Their respective absorbance was measured in a spectrophotometer at 260nm using reagent blank to calibrate the instrument at zero. The tannin content was calculated using the formula, 41

% Tannin = An/W x C/Va x Vf x 100/1

Where:

An = absorbance of test sample, AS = absorbance of standard solution, C = concentration of standard solution, W = weight of sample used, Vf = total volume of extract, Va =volume of extract analyzed.

3.6.3 DETERMINATION OF OXALATE The titration method (AOAC, 2006) was used. Two grams of sample was suspended in a mixture of 190ml of distilled water in a 250ml volumetric flask. 10ml of 6M HCl and the suspension heated for 1 hour at 100oC in a water bath. The mixture was cooled and made up to 250ml mark with distilled water before filtration. Duplicate portion of 125ml of the filtrate was measured into 250ml beakers. Each extract was made alkaline with concentrated sodium then made acid by drop wise addition (4 drops) of acetic acid until the test solution is changed from salmon pink to faint yellow (pH 4-4.5) (methyl red indicator used). Each portion was heated at 90oC to remove precipitate containing ferrous ions. The filtrate was heated again to 90oC on a hot water bath and 10ml and 5% calcium chloride solution added while being stirred constantly. After heating, it was centrifuged at full speed (2500 rpm) for 5minutes. The supernatant was decanted and the precipitate completely dissolved in 10ml of

20% (v/v) H2SO4 solution and the total filtrate resulting from 2g of the sample was made up to 300ml.

Permaganate titration: Aliqout 125ml of the filtrate was heated until near boiling and then titrated against 0.05M KMNO4 solution to a faint pink colour which persisted for 30 seconds. Oxalic acid content was calculated using the formula,

5 %Oxalic acid = T x (Vme) (Df) x 10 ME x Mf where, T = Titre of KMNO4 (ml), Vme = volume - mass equivalent (1ml of 0.05M MNO4 solution is equivalent to 0.0022g anhydrous oxalic acid), Df = the dilution factor (i.e 300ml)

125ml, ME = the molar equivalent of KMNO4 in oxalic acid (KMNO4 redox reaction is 5),

Mf = the mass of the sample used.

3.6.4 DETERMINATION OF HEMAGGLUTININ Hemagglutinin determination was by spectrophotometric method as described by Onwuka (2005). Furthermore, 0.5g of the sample was weighed and dispersed in 10ml normal saline 42 solution buffered at pH 6.4 with a 0.01M phosphate buffer solution. This was allowed to stand at room temperature for 30minutes and then centrifuged to obtain the extract. To 0.1ml of the extract diluents in the test tube 1ml of trypsinized albino rat blood was added. The control was mounted with the test tube containing only the red blood cells. Both tubes were allowed to stand for 4hours at room temperature. 1ml of normal saline was added to all the test tubes and allowed to stand for 10minutes after which the absorbance was read at 620nm. The test tube containing only the red blood cells and normal saline served as the blank. The result was expressed as Hemagglutinin units per milligram of the sample.

Hemagglutinin unit/mg = (b-a) x F

Where b= absorbance of test sample solution, a = absorbance of the blank control,

F= experimental factor given by

F= (1/w x Vf / Va) D

Where w= weight of sample, Vf = total volume of the extract, Va = volume of the extract used in the assay, D = dilution factor (1ml to 10ml and 0.1ml out of 10ml) i.e 100.

3.7.0 DETERMINATION OF MINERAL CONTENT The mineral content of the formulated samples were evaluated using the method described by Adedeye and Adewoke (1992). One gram of dried samples was digested with 2.5ml of 0.03N hydrochloric acid (HCl). The digest was boiled for 5 minutes, allowed to cool to room temperature and transferred to 50ml volumetric flask and made up to the mark with diluted water. The resulting digest was filtered with ashless Whatman No. 1 filter paper. Filtrate from each sample was analyzed for mineral (calcium, phosphorus, magnesium, Iron, sodium, manganese, copper and zinc) contents using an Atomic Absorption Spectrophotometer (Buck Scientific Atomic Absorption Emission Spectrophotometer model 205, manufactured by Nowalk, Connecticut, USA) using standard wavelengths. The real values were extrapolated from the respective standard curves. Values obtained were adjusted for HCl-extractability for the respective ions. All determinations were performed in triplicates.

3.8.0 DETERMINATION OF VITAMIN CONTENT

3.8.1 DETERMINATION OF VITAMIN B1 (THIAMINE) Thiamin was determined using AOAC (2006) procedure. A 75 ml of 0.2 N HCl was added to 2g of sample and the mixture boiled over a water bath. After cooling, 5ml of phosphatase 43 enzyme solution was then added and the mixture incubated at 37oC overnight. The solution was placed in 100ml volumetric flask and the volume made up with distilled H2O. The solution was then filtered and the filtrate purified by passing through silicate column. To 25ml of the filtrate in a concical flask was added 5ml acidic KCl eluate, 3 ml of alkaline ferricyanide solution, and 15 ml isobutanol, and shaken for 2min. The solution was allowed to separate and the alcohol layer taken. About 3g of anhydrous sodium sulphate was added to the alcohol layer. A 5 ml of thiamine solution was accurately measured into another 50 ml stoppered flask. The oxidation and extraction of thiochrome as already carried out with the sample was repeated using the thiamin solution. A 3ml of 15% NaOH was added to the blank instead of alkaline ferricyanide. The blank sample solution was poured into fluorescence reading tube and reading taken at the expression: % thiamin = X/Y x 1/5 x 25/V x 100/W

Where W = weight of sample, X = reading of sample – reading of blank, Y = reading of thiamin standard –reading of blank standard, V = volume of solution used for test on the column.

3.8.2 DETERMINATION OF VITAMIN B2 (RIBOFLAVIN) AOAC (2006) standard method was used. A 2 g portion of each of the formulated samples was placed in a conical flask and 50 ml of 0.2 N HCl added .The solution was boiled for 1 hour, and cooled. The pH was adjusted to 6.0 using sodium hydroxide. A 1 N HCl was added to the sample solution to lower the pH to 4.5. The solution was then filtered into 100 ml volumetric flask and made up to volume with distilled water. In order to remove interference, two tubes were taken and labeled 1 and 2. About 10 ml of water was added to tube 1. Another 10 ml of filtrate and 1 ml riboflavin standard was added to test tube 2. A 1 ml of glacial acetic acid was added to each tube and mixed. Then, 0.5 ml 3% KMnO4 solution was added to each tube. The test tube was allowed to stand for 2 min, after which 0.5 ml 3% H2SO4 was added and solution mixed well. The flourimeter was adjusted to excitation wavelength of 470nm and emission wavelength of 525nm. The flourimeter was also adjusted to zero deflection against 0.1 N H2SO4 and 100 against tube 2 (standard).The fluorescence of tube 1 was added to both tubes and the fluorescence measured within 10 sec. Riboflavin was then calculated as

Riboflavin mg/g = Y/Y-X x 1/W

Where W = weight of sample, X = reading of sample – blank reading, 44

Y = reading of sample + standard (tube 2)- reading of sample - standard blank.

3.8.3 DETERMINATION OF VITAMIN B6 (PYRIDOXINE)

AOAC (2006) method was used in determining vitamin B6. A 2 g portion of each of the formulated samples was weighed into 500 ml Erlenmeyer flask and 200 ml 0.4 M HCl added. The solution was autoclaved for 2 h at 1210C, cooled to room temperature and pH adjusted to 4.5 with 6M KOH. The solution was diluted to 250 ml with water in volumetric flask and filtered through Whatman No. 40 paper. A 40–200 ml filtered aliquot was taken for chromatography. Desired amount of the filtered extract was placed on ion exchange column in 50 ml portions and allowed to pass completely through with no flow regulation. Beaker and column were washed 3 times with 5 ml portions hot 0.02 CH3COOK (pH 5.5). Pyridoxal was eluted with two 50 ml portion boiling 0.04 M CH3COOK (pH 6.0) using 100 ml volumetric flask as receiver. Pyridoxine was eluted with two, 50ml portions boiling 0.1 M

CH3COOK (pH 7.0), using 100 ml volumetric flask as receiver. Pyridoxamine was eluted with two 50 ml boiling KClK2HPO4 (pH 8.0) solution, using 250 ml beaker as receiver and the pH adjusted to 4.5. Pyridoxine and pyridoxal eluates were diluted to 100 ml and pyridoxamine to 200 ml with water. A 10 ml each of the standard pyridoxine, pyridoxal and pyridoxamine solution was then neutralized with KOH and adjusted pH 4.5 with CH3COOH. The resulting solutions were each put on column, washed and eluted as above. Eluted pyridoxine and pyridoxal standards were diluted to 100 ml and pyridoxamine to 200 ml with water. Each standard was diluted to 1.0 mg/ml with water.

Assay: Clean tubes and glass beads were heated at 2600C for 2 hours. Two 4 mm glass beads were placed in each 16 x 150mm screw-cap glass culture tube. For standard curve, freshly prepared standard working solutions was pippetted into triplicate tubes to give 0.0, 0.1, 2.0, 3.0, 4.0, and 5.0ng pyridoxine, pyridoxal, or pyridoxamine/tube respectively. Similarly test tubes for eluted standards were prepared, omitting blanks. Test eluates from chromatographic column were diluted to contain 1ng vitamin B6 component/ml 1,2,3,4 and 5 ml diluted eluates were pipepetted into triplicate tubes. Tubes were capped with plastic caps with 3 mm (1/8 inch) hole through top. Entire set were autoclaved for 10 min at 1210C and cooled to room temperature. Using automatic pipette with sterilized attachments, 5ml steamed medium (previously prepared) was pipetted through hole in the cap. Tubes were covered with sterile cheese cloth and placed in refrigerator for 1 hour followed by inoculation. Aseptically, 1 drop assay inoculum of S. uvarum suspended cells was inoculated through cap of each tube, except for first set of 0.0 level standard curves. Tubes were then inoculated on constant rotary shaker 45

22 hours in a temperature-regulated room (30 h).Tubes were steamed in an autoclave for 5 minutes, cooled, and the caps removed. % T at 550nm was read on spectrophotometer. 100% T was set with water to read inoculated blank. 100% T was set with un- inoculated blank to read inoculated blank. Nine inoculated blank tubes were mixed, and with this mixture set at 100% T on instrument, all other tubes were read. Readings in triplicate tubes were averaged and % T plotted against ng eluted standard pyridoxine, pyridoxal, or pyridoxamine/tube was determined by interpolation and µg pyridoxine, pyridoxal and pyridoxamine /g sample reported.

3.8.4 DETERMINATION OF VITAMIN B12 AOAC (2006) method was used in determining vitamin B12. 1g of each sample was weighted into a 250ml volumetric flask. 100ml of distilled water was added and spanned or shaken for 45min and made up to mark with distilled water. The sample mixture was filtered into another 250ml beaker, rejecting the first 20mls that had been filtered. Another 20ml filtrate was collected. To the filtrate, 5ml of 1% sodium dithionite solution were added to decolourized the yellow colour. Standard cyanocobalamin of range 0 -10 ug/ml were prepared from stock cyanocobalamin. A sample blank made up to distilled water was also prepared. The absorbance of samples as well as standard were read at a wavelength of 445nm on a spectronic 21D spectrophotometer. Vitamin B12 (cyanocobalamin) = Absorbance of sample x Gradient Factor x Dil. Factor Wt. of sample

3.8.5 DETERMINATION OF VITAMIN C (ASCORBIC ACID) Ascorbic acid was determined according to the 2, 6 – dichlorophenol titermetric method of AOAC (2006). A 2g of the sample was homogenized with acetic acid solution and extracted. Vitamin C standard solution was prepared by dissolving 50 mg standard ascorbic acid tablet in 100ml volumetric flask with distilled water. The solution was filtered out and 10 ml of the clear filtrate added into a conical flask in which 2.5 ml acetone had been added. This was titrated with indophenol dye solution (2,6 - dichlorophenol indophenol) for 15 seconds. The procedure was followed for the standard as well. Ascorbic acid was calculated as:

Ascorbic acid (m/g) sample = C x V x (DF/WT)

Where C = mg ascorbic acid/ml dye, V = volume of dye used for titration of diluted sample DF = Dilution factor, WT = weight of sample (g)

3.9.0 DETERMINATION OF ESSENTIAL AND NON-ESSENTIAL AMINO ACIDS The method used for the essential and non-essential amino acids was as described by AOAC (2006). 20μg of each of the formulation was dried in conventional hydrolysis tubes. To each tube 100μL of 6mol L-1 HCl containing 30ml phenol and 10ml 2-mercaptoethanol (6mol L-1 HPME) were added and the tubes were evacuated, sealed and hydrolyzed at 110oC for 22hours. After hydrolysis, HCl was evaporated in a vacuum bottle heated to about 60oC. The residue was dissolved in a sample buffer and analyzed for amino acids using RP-HPLC with an Agilent 1100 assembly system (Agilent Technologies, Palo Alto, CA 94306, USA) and Zorbax 80A C18 column (4.6 id x 180 mm). The Excitation Wavelength (Ex) of 348 nm and Emission Wavelength (Em) of 450 nm were chosen. The column oven was maintained at 60oC. Amounts of amino acids were determined by calculations using the recorded chromatogram. For cystine determination, 50μg of the formulations were first oxidized with 10μl performic acid in an ice-water bath for 4 hours. The mixtures were evaporated with a vacuum pump to remove performic acid before hydrolysis. Determination of tryptophan was done by the ninhydrin method. One gram of each formulation was put into a 25ml polypylene test tube with caps, 10ml of 0.075 N NaOH was added and thoroughly mixed until clear solution was obtained. The dispersion was shaken for 30 min and centrifuged at 5000rpm for 10 min and the supernatant liquid transferred into a clean test tube. 0.5mL of the supernatants, 5ml of ninhydrin reagent (1.0g of ninhydrin in 100 ml mixture of 37% HCl and 96% HCOOH) in a ratio of 2:3 for all the samples were added and incubated at 35oC for 2hours. After incubation, the solution was cooled to room temperature (23-25oC) and the volumes were made up to 10ml using diethyl ether, thoroughly mixed using a vortex mixer, filtered and the clear filtrates were analyzed with the same equipments as described above for the other amino acids.

3.10. MICROBIOLOGICAL EXAMINATION Microbiological analysis was carried out using the pour plate method as described by Onwuka (2005). Total viable bacteria, molds and coliform counts were estimated by multiplying the means of the total colonies by the dilution factor.

DATA ANALYSIS: The experiment was conducted in a completely randomized design (CRD). Data obtained were subjected to one-way analysis of variance (ANOVA) and mean separation was done by Duncan multiple range test (p=0.05), using Statistical Package for Social Sciences (SPSS) version 17.0. 47

CHAPTER FOUR 4.0 RESULTS AND DISCUSSION

4.1 PROXIMATE COMPOSITION The mean values of the proximate composition of the formulated samples are as shown in Table 7. The results revealed some significant changes at p<0.05.

4.1.1 MOISTURE The moisture content ranged from 3.38+0.01 to 4.2+0.01%, with the highest value observed in the breakfast cereal containing 50:50 formulations. This is probably due to the high content of coconut fiber that has the ability to imbibe moisture from the environment and swell. Coconut has been shown to have hygroscopic or water-absorbing properties (Wasserman, 2010). The low moisture content generally observed in the samples may add the advantage of prolonging the shelf life of the products, if properly packaged.

4.1.2 PROTEIN The protein content of the samples ranged from 15.68+0.07% to 18.26+0.13%. These values are higher than other related previous studies; lower values were recorded for the commercial control sample, Weetabix Original (11.50%), a breakfast meal containing AYB, maize, sorghum and soybean (13.53+1.83-15.02+2.30%) (Agunbiade and Ojezele, 2010) as well as breakfast cereal made from treated pigeon pea and sorghum (Mbaeyi, 2005) respectively. The high protein content of the products may be attributed to the presence of African yam bean (AYB) flour used in the formulations. Raw AYB has been reported to contain about 20-23% protein (Obatolu et al., 2001). The progressive solubilization and leaching out of the nitrogenous substances during soaking and boiling of the legume may be responsible for the slight protein reduction in the samples (Ukachukwu and Obioha, 2000) other than these. The generally high level of protein, however demonstrates the effect of supplementing legumes in breakfast cereals.

Table 8: Proximate Composition of Breakfast Cereals from Blends of AYB +Maize: Defatted Coconut flour

Sample Moisture Protein Fat Ash Crude Fiber Carbohydrate (%) (%) (%) (%) (%) (%) 100:0 3.38+0.02e 18.26+0.13a 1.84+0.02d 5.29+0.02f 6.70+1.80b 64.53+0.05a 90:10 3.54+0.02d 17.98+0.09b 1.91+0.02c 5.59+0.01e 8.57+0.01a 62.41+0.41a 80:20 3.81+0.01c 17.69+0.06c 1.98+0.01b 5.87+0.01d 8.68+0.02a 61.97+0.09a 70:30 4.04+0.01b 17.62+0.06c 1.99+0.03b 5.96+0.01c 8.81+0.01a 61.58+0.16a 60:40 3.99+0.08b 17.19+0.06d 1.99+0.12a 6.86+0.05b 9.01+0.01a 60.96+1.42b 50:50 4.20+0.01a 15.68+0.07f 2.02+0.02a 7.36+0.02a 9.08+0.07a 61.66+1.15a

Values are means +SD of triplicate determinations Means differently superscripted along the vertical columns are significantly different (p<0.05) Sample ratio - AYB+ Maize flour: defatted coconut flour.

4.1.3 FAT The results of the analysis show that the fat content of the formulated breakfast cereals were generally low, ranging from 1.84+0.02% to 2.02+0.02%. This range of values agrees with that recorded for the control sample- Weetabix (2.00%). Significant differences (p<0.05) were observed among the samples. The presence of graded levels of defatted coconut fiber in the formulations may be responsible for the generally low fat content of the resulting products, although most of the legumes, with the exception of groundnuts and soybeans contain less than 3% fat (Ihekoronye and Ngoddy, 1985). Higher fat values were recorded for fortified breakfast cereals made from AYB, maize, sorghum and soybean as 3.7+0.36% (Agunbiade and Ojezele, 2010) and breakfast cereals made from Sorghum and Pigeon pea composite flour as 8.70- 14.2% (Mbaeyi, 2005). The low fat content of the developed products would be suitable for weight watchers.

4.1.4 ASH The results of the ash content analysis of the formulated samples showed significant differences (p<0.05) with values ranging from 5.29+0.02 to 7.36+0.02%. Lower values, 1.36+0.05% (Agunbiade and Ojezele, 2010) and 1.50-2.50% (Mbaeyi, 2005) were recorded by other researchers. The high ash values recorded in this work may be attributed to the presence of defatted coconut fiber and whole maize grains used as part of the ingredients in this study. Coconut fiber belongs to the class of compounds known as flammable solids. It easily catches fire upon ignition, thus producing more ash on combustion (Wasserman, 2010).

4.1.5 CRUDE FIBER The values obtained from the determination of crude fiber content of the formulated breakfast cereals ranged from 6.70+1.80% to 9.08+0.07%. Lower values, 3.1- 3.8% (Agunbiade and Ojezele, 2010) and 1.54- 4.0% (Mbaeyi, 2005) were previously recorded for other breakfast cereals formulation. The control sample- Weetabix however contained a fiber value of 10%. Fiber is needed to assist in digestion and keep the gastrointestinal tract healthy and can also help to keep the blood sugar stable. It slows down the release of glucose during digestion, so cells require less insulin to absorb that glucose. The American Diabetes Association recommends that people with diabetes should consume 25-50g of fiber per day (Trinidad et al., 2006). The fecal bulking action of insoluble fiber makes it useful in the treatment of constipation and diverticular disease (McKevith, 2004).

4.1.6 CARBOHYDRATE The values from the carbohydrate content analysis of the formulated samples ranged from 60.96+1.42 to 64.53+0.05%. Apart from the sample containing 60:40 formulation, all other samples were not significantly different (p<0.05). Higher carbohydrate values were reported for breakfast cereals formulated from sorghum and pigeon pea (Mbaeyi, 2005) as well as the control- Weetabix (68.4%). The higher carbohydrate values recorded by other researchers may be attributed to the high content of the cereals and legumes used as the principal ingredients in the formulations (Kanu et al., 2009).

4.1.7 ENERGY The values obtained for the total energy content of the formulated samples shown in Figure 7, ranged from 327.54 to 347.72Kcal and were found to be within the range of values recorded for breakfast cereals made from treated and untreated sorghum and pigeon pea (316.46- 420kcal) as well as treated ready-to-eat breakfast cereals (314 - 420kcal) (Mbaeyi, 2005; Kent, 1983). Similar value was also recorded for the control sample- Weetabix as 338kcal. These values represent the amount of energy in food that can be supplied to the body for maintenance of basic body functions such as breathing, circulation of blood, physical activities and thermic effect of food. Increasing addition of coconut fiber was inversely proportional to the energy value of the products. 51

Figure 7: Energy value of Breakfast Cereals made from blends of AYB + Maize: Defatted Coconut flour

LEGEND:

A= 100:0 B=90:10 C=80:20 D=70:30 E=60:40 F=50:50 Sample - AYB+ Maize flour: defatted coconut flour.

4.2.0 FUNCTIONAL PROPERTIES The result of evaluation of the functional properties of the developed breakfast cereals is shown in Table 8.

4.2.1 pH The pH values of the products which ranged from 4.70+0.01 to 6.56+0.01 showed that there were no significant differences (p>0.05) between samples containing 70:30, 60:40 and 50:50 formulations, as well as between samples 90:10 and 80:20 formulations, while there was significant difference (p<0.05) in the pH of 100:0 formulation and the other samples. Agunbiade and Ojezele (2010) recorded slightly lower values (4.88) for fortified breakfast cereal made from maize, sorghum, AYB and soybeans. The pH range observed in this study may be due to partial hydrolysis which might have occurred during soaking of the legume. The higher pH values recorded for the samples with high level of defatted coconut fiber (20- 50%) may be as a result of its composition.

4.2.2 BULK DENSITY The results of bulk density of the breakfast cereals ranged from 0.29+0.01g/ml to 0.71+0.01g/ml with the highest value found in the sample with 100:0 formulation. There was a gradual reduction of the bulk density with increase in the addition of defatted coconut flour content although the samples with 90:10, 80:20, 70:30 formulations did not have significant differences (p>0.05). Higher values of bulk density (2.45+0.10 and 2.60+0.05) were recorded by Agunbiade and Ojezele (2010) for fortified breakfast cereals made from maize, sorghum, AYB and soybeans. However, Mbaeyi (2005) recorded values that were similar to those obtained in this study (0.5341- 0.7267g/ml). The bulk densities of the product may require identical packaging space. The less the bulk density, the more packaging space is required (Agunbiade and Ojezele, 2010).

4.2.3 WATER ABSORPTION CAPACITY (WAC) The results obtained for water absorption capacity of the formulated breakfast cereals ranged from 68.31+0.01 to 76.39+0.01%. It was found to increase with increase in defatted coconut flour inclusion. This may be connected to the fact that coconut fiber has hygroscopic properties, thus, swelling on exposure to moisture (Wasserman, 2010). Similar values were obtained from treated and untreated sorghum and pigeon pea breakfast cereals (Mbaeyi, 2005). 53

4.2.4 OIL ABSORPTION CAPACITY The oil absorption capacity (FAC) of the breakfast cereals varied in trend from those obtained for water absorption capacity. The values ranged from 0.87+0.01to 1.32+0.01% with the highest value recorded for the sample with 100:0 formulation. The hydrophobicity of proteins is known to play a major role in fat absorption. This acts to resist physical entrapment of oil by the capillary of non-polar side chains of the amino acids of the protein molecules (Chau and Cheung, 1998). There were significant differences (p<0.05) among all the samples. The FAC decreased with increasing addition of defatted coconut flour.

4.2.5 FOAM CAPACITY The foam capacity of the samples ranged from 2.48+0.01 to 3.49+0.01% with the highest value observed in the sample with 100:0 formulation. There was a gradual decrease in foam capacity with increasing addition of defatted coconut flour. This value is higher than those recorded for flour obtained from boiled AYB (1.98%). Padmashree et al. (1987) also reported the decreasing effect of processing conditions on foam capacity with processed cowpea flour. The more pronounced reduction in foam capacity in heat-treated (boiling and roasting) sample has been attributed to protein denaturation (Lin et al., 1974). It is also an indication of precipitation of proteins due to temperature and some heat treatment.

4.2.6 VISCOSITY The viscosity of the products ranged from 19.73+0.01 to 31.08+0.01cps, and it was the sample with 50:50 formulation that had the least value. The generally low viscosity observed may be due to less disruption of intermolecular hydrogen bonds that brought about noticeable swelling of the granules and gelation (Iheoronye and Ngoddy, 1985). Swelling of the granules was observed to be slight in cold water. According to Wasserman (2010), coconut fiber has a high water absorption capacity and easily dissolves in liquids, but does not thicken or gel.

4.2.7 IN-VITRO PROTEIN DIGESTIBILITY The results obtained for the invitro-protein digestibility shown in Figure 8, ranged from 66.30+0.01 to 82.2+0.01%. The sample with 50:50 formulation had the highest digestibility value. This shows that more protein was digested with the presence of more coconut fiber. This may be connected to the fact that fiber is known to aid digestion, and this might have led to the increase in digestibility of the proteins. The in-vitro protein digestibility has been 54 reported to be affected by many factors such as genotype and tannin content (Elsheikh et al., 1999).

4.2.8 GELATION CAPACITY The gelation capacity of the formulated samples varied from 75.32+0.01 to 89.66+0.01% with the highest value found in the sample with 100:0 formulation. A gel can represents a transitional phase between solid and liquid states. In food systems, the molecular net consists of proteins, polysaccharides or a mixture of both, while the liquid is usually water. Ionic strength, pH and the presence of non-protein components can influence the gelation properties (Sridaran and Karim, 2011). The gradual reduction in the gelation capacity with increasing defatted coconut ratio may be as a result of high fiber content which is known to have a high water absorption capacity and thus does not thicken or gel on heating (Wasserman, 2010).

Table 9: Functional Properties of Breakfast Cereals from Blends of AYB+Maize: Defatted Coconut Flour

Sample pH BD WAC FAC FC Viscosity GC (g/ml) (%) (%) (%) (cps) (%)

100:0 4.70±0.01c 0.30±0.01a 68.32±0.01f 1.32±0.01a 3.49±0.01a 31.08±0.01a 89.66±0.01a

90:10 5.27±0.01b 0.26±0.01b 70.29±0.01e 1.13±0.01b 3.43±0.01a 30.56±0.01a 84.29±0.01b

80:20 5.30±0.01b 0.24±0.01b 71.24±0.01d 1.07±0.01c 3.25±0.01ab 26.41±0.01b 81.4±30.01c

70:30 6.23±0.01a 0.24±0.02b 74.81±0.05c 0.96±0.01d 2.80±0.01b 24.22±0.01c 78.56±0.01d

60:40 6.55±0.01a 0.19±0.01c 75.43±0.01b 0.93±0.01e 2.63±0.01e 21.98±0.01d 77.34±0.01e

50:50 6.56+0.01a 0.17+0.01d 76.39+0.06a 0.87+0.01f 2.48+0.01f 19.73+0.01e 75.32+0.01f

Values are means +SD of triplicate determinations Means differently superscripted along the vertical columns are significantly different (p<0.05) Sample ratio: AYB+ Maize: Defatted coconut fiber

Figure 8: In-vitro protein digestibility of breakfast cereals made from blends of AYB+Maize: coconut fiber LEGEND:

A= 100:0 B=90:80 C=80:20 D=70:30 E=60:40 F=50:50 Sample ratio: AYB+Maize: Defatted coconut flour

4.3.0 SENSORY EVALUATION The mean sensory scores obtained from the formulated samples are shown in Tables 10-13. The results were recorded in four groups according to the way they were served to the panelists. The groups included: i. Samples served dry (as it is). ii. Sample served with cold water (added to a bowl of cold water). iii. Sample served with cold water and milk (Peak instant full cream powder). iv. Sample served with hot water and milk (Peak instant full cream powder).

4.3.1 ATTRIBUTE PERCEPTION OF THE SAMPLES SERVED DRY The result obtained from serving the samples obtained as they were (dry) to the assessors is presented in Table 10. It shows that there were no significant (p>0.05) differences between the samples in all the attributes evaluated, except the control that was significantly different (p<0.05) in terms of appearance, flavor, taste, consistency and overall acceptability. In terms of consistency, the sample with 70:30 formulation ranked next to the control sample (Weetabix), while samples with 90:10 and 50:50 formulations showed closest similarities to the control sample in terms of flavour. The reason for this may be attributed to the strong AYB and coconut flavours which were observed to be outstanding in these samples, thus comparing well with the control sample. In terms of taste, the sample with 70:30 formulation ranked next to the control sample, although it showed no significant (p>0.05) difference with other samples. In terms of aftertaste, the judges preferred the samples containing 90:10 and 50:50 formulations along with the control. This also may be due to the strong taste and flavor in the AYB and defatted coconut prominent in these samples, which lingered in the mouth after swallowing. It is also an indication that the processing technique employed in the production of the formulated samples was able to significantly reduce the beany flavor inherent in AYB, thus making the products desirable.

In terms of mouthfeel, sample ratios 100:0 and 60:40 were significantly different (p<0.05) from the control and the rest. In terms of overall acceptability, none of the samples was rejected by the assessors; however the commercial control was the most acceptable probably because the assessors were accustomed to the product, then followed by sample ratios 70:30, 50:50, 100:0, 90:10, 80:20 and finally 60:40.

Table 10: Mean Sensory Scores for Formulated Breakfast Cereals Served Dry

Sample Appearance Consistency Flavour Taste Aftertaste Mouthfeel Overall Acceptability

100:0 5.73+ 1.79a 6.00+1.36b 5.27+1.27b 5.60+1.76b 5.00+1.65b 5.60+1.24b 5.93+1.16b

90:10 6.73+0.79a 6.07+0.59b 6.00+1.00b 5.87+0.99b 5.93+1.22ab 6.00+1.07ab 5.87+1.25b

80:20 6.13+1.13a 5.93+0.88b 5.60+1.12b 5.67+1.04b 5.47+1.19b 5.80+0.94ab 5.67+1.23b

70:30 6.53+1.30a 6.07+1.33b 5.67+1.17b 6.07+1.28b 5.53+1.25b 5.80+1.01ab 6.13+1.36b

60:40 6.13+1.01a 5.80+1.42b 5.40+1.35b 5.33+1.49b 4.93+1.33b 5.40+1.35b 5.47+1.36b

50:50 6.27+1.49a 5.53+1.81b 5.93+1.16ab 5.87+1.12b 6.00+1.13ab 5.53+1.13ab 6.00+0.85b

Weetabix 5.67+2.06a 7.20+0.94a 6.87+1.41a 7.07+1.22a 6.67+1.50a 6.53+1.55a 7.13+1.19a Values are means +SD of triplicate determinations Means differently superscripted along the vertical columns are significantly different (p<0.05)

Sample ratio: AYB+ Maize: Defatted coconut fiber

4.3.2 ATTRIBUTE PERCEPTIONS OF THE SAMPLES SERVED WITH WATER The results of the sensory scores of the samples served by placing the samples in a bowl of water at room temperature (t = 28±2°C) is shown in Table 11. Addition of water altered the assessors‟ perception of the samples‟ attributes. The samples and the control were not significantly different (p>0.05) from each other in terms of flavor, taste, aftertaste, mouthfeel and overall acceptability. This may be attributed to dissolution of the samples, which neutralized some of the attributes by the water used to serve the samples. In terms of appearance the samples with 70:30, 60:40 and 50:50 formulations were most preferable. Their scores were significantly higher (p<0.05) than other samples including the control. In terms of consistency, all the samples, except that with 100:0 showed no significant difference (p>0.05) from the control. Consuming the samples in water reduced the differences in the ratings between the samples and the control. The fact that the samples had closer attributes shows that the formulated samples have the potential of being acceptable when introduced to consumers.

Table 11: Mean Sensory Scores for Samples Served with Cold Water

Sample Appearance Consistency Flavour Taste Aftertaste Mouth feel Overall Acceptability

100:0 5.25+2.08ab 5.13+2.42b 6.79+2.12a 6.06+2.17ab 5.12+2.21a 5.25+2.49ab 5.44+2.42a

90:10 5.80+2.21ab 6.07+1.94ab 5.27+2.34a 5.07+2.22b 6.33+2.09a 4.80+2.14ab 5.60+2.26a

80:20 5.67+2.06ab 5.80+2.24ab 6.00+2.07a 5.27+1.94ab 5.20+2.08a 4.60+1.99b 5.53+2.20a

70:30 6.53+1.60a 5.60+2.10ab 6.60+1.45a 6.20+1.97ab 5.60+2.50a 6.00+1.77ab 5.53+1.99a

60:40 6.47+2.03a 6.13+1.92ab 6.13+1.92a 5.27+1.88ab 5.67+1.80a 5.67+1.84ab 6.00+1.51a

50:50 5.81+2.16a 5.57+2.79ab 6.18+2.09a 6.86+1.87a 6.36+2.09a 6.36+1.95ab 6.07+2.89a

Weetabix 4.40+1.99b 7.13+2.26a 6.47+2.26a 6.33+2.69ab 6.40+2.82a 6.47+2.89ab 6.47+2.77a

Values are means +SD of triplicate determinations Means differently superscripted along the vertical columns are significantly different (p<0.05) Sample ratio: AYB+ Maize: Defatted coconut fiber

4.3.3. ATTRIBUTE PERCEPTION OF THE SAMPLES SERVED WITH COLD MILK

The mean sensory scores of serving the samples with cold milk shown in Table 12, revealed that there were significant differences (p<0.05) between the samples and the control in all the attributes except appearance, probably because of the masking effect of the colour of the samples by milk. In terms of consistency, the samples with 100:0 and 90:10 formulations ranked next to the control. This may be as a result of the low content of defatted coconut fiber that visibly improved uniformity of these samples, and enhanced dissolution of the samples into tiny particles, which made them more desirable. The sample with 50:50 formulation scored the least mark, which may be related to the high fiber in the sample making it less homogenous. In terms of flavour, significant changes (p<0.05) were observed in all the samples; however the control shared some similarities with the samples containing 100:0 and 90:10 formulations. These however had some similarities with samples containing 80:20 and 70:30 formulations. The samples with 60:40 and 50:50 formulations attracted least scores, which may be due to the high level of defatted coconut fiber present in them, thus masking all other ingredients. In terms of taste and aftertaste, significant differences (p<0.05) were observed between the samples and the control which had the highest score, while samples with 60:40 and 50:50 formulations were scored least. This again may be due to the higher percentage of the defatted coconut fiber present in these samples that may have masked all other ingredients, thereby altering their taste. In terms of mouthfeel and overall acceptability, all the samples were preferred next to the control, except 50:50 formulation that was least acceptable.

Table 12: Mean Sensory Scores for Formulated Breakfast Cereals served with cold Milk

Sample Appearance Consistency Flavour Taste Aftertaste Mouthfeel Overall Acceptability

100:0 6.40+1.30a 6.00+1.31b 6.40+0.99ab 6.77+1.36b 6.07+1.22b 6.00+1.13b 6.00+1.31bc

90:10 6.60+0.83a 5.93+0.96b 6.27+0.70ab 6.20+0.94bc 5.80+1.08b 6.00+1.13b 6.13+0.74b

80:20 6.13+1.19a 5.73+0.96bc 5.53+1.13bc 5.73+1.22bcd 5.47+1.13b 5.40+0.99bc 5.80+0.94bc

70:30 6.33+0.98a 5.80+1.21bc 5.80+1.42bc 5.60+1.55bcd 5.40+1.35b 5.20+1.37bc 5.53+1.46bc

60:40 6.20+1.01a 5.47+0.99bc 5.13+1.06c 5.20+1.15d 5.13+1.30b 5.07+1.22bc 5.13+1.25b

50:50 5.87+1.13a 5.07+0.96c 5.33+1.45c 5.27+1.09cd 5.20+1.26b 4.67+1.23c 5.13+0.92c

Weetabix 5.93+1.98a 7.40+0.83a 7.00+1.07a 7.33+0.89a 7.07+1.09a 7.33+0.98a 7.47+0.83a

Values are means +SD of triplicate determinations Means differently superscripted along the vertical columns are significantly different (p<0.05) Sample ratio: AYB+ Maize: Defatted coconut fiber

4.3.4. ATTRIBUTES PERCEPTION OF THE SAMPLES SERVED WITH HOT MILK

The mean sensory scores presented in Table 13 shows the influence of serving the samples with warm milk (50°C), which altered the attributes perception of the samples compared to the samples served with cold milk. The commercial control sample was the most preferred in all the attributes except appearance that the sample with 90:10 formulation was most preferred, and significantly different (p<0.05) from the control sample, which was least preferred, probably because of its darker colour compared to the formulated samples. In terms of consistency, all the formulated samples however showed no significant difference (p>0.05) among them. In terms of flavor, samples with 100:0 and 90:10 formulations were not significantly (p>0.05) different from the control. These two samples shared similarities with the sample containing 70:30 formulation and then with the other samples. In terms of taste, the samples with 100:0, 90:10, 70:30 formulations were preferred alongside the control which were significantly different (p<0.05) from other formulated samples that shared similar characteristics. In terms of aftertaste the samples with 100:0, 90:10 and 70:30 formulations showed no significant (p>0.05) difference with the control. The samples with 80:20, 60:40 formulations were scored below average but shared similarities with the samples containing 100:0 and 50:50 formulations respectively. In terms of mouth feel, only the sample with 90:10 formulation shared some similarities with the control. This sample also shared some similarities with samples containing 100:0 and 70:30 formulations, but was significantly different (p<0.05) from samples with 80:20, 60:40, 50:50 formulations. All the samples except those containing 60:40, 50:50 formulations scored above average. In terms or overall acceptability, it was observed that the samples with 90:10 formulation shared some similarities with the control as well as other samples except that with 60:40 formulation.

Table 13: Mean Sensory Scores for Samples Served with Hot Milk (50°C)

Sample Appearance Consistency Flavour Taste Aftertaste Mouthfeel Overall Acceptability

100:0 6.53±1.36ab 5.67±1.40b 5.73±1.51bc 5.47±1.36abc 5.40±1.45ab 5.33±1.35bc 5.80±1.36bc

90:10 6.80±0.68a 6.13±0.92b 6.20±0.86ab 6.33±0.89ab 5.87±0.92ab 6.13±0.83ab 6.20±0.77ab

80:20 6.00±1.07ab 5.53±1.25b 5.00±1.25c 5.33±1.45bc 4.73±1.16c 5.00±1.25c 5.47±1.36bc

70:30 6.53±0.83ab 5.67±1.40b 5.53±1.51bc 5.47±1.36abc 5.40±1.45ab 5.33±1.35bc 5.47±1.36bc

60:40 6.33±0.97ab 5.27±0.80b 4.73±1.33c 4.73±1.16c 4.40±1.30c 4.67±0.98c 5.07±1.10c

50:50 6.27±1.28ab 5.27±1.39b 5.73±1.36abc 5.27±1.62bc 5.00±1.65bc 4.87±1.46c 5.33±1.20bc

Control 5.73±1.94b 7.40±1.01a 6.67±1.45a 6.53±1.68a 6.40±1.76a 6.87±1.88a 6.87±1.81a

Values are means +SD of triplicate determinations Means differently superscripted along the vertical columns are significantly different (p<0.05) Sample ratio: AYB+ Maize: Defatted coconut fiber

4.3.5 EFFECT OF SERVING STYLE ON SENSORY ATTRIBUTES OF THE SAMPLES (AYB+ Maize: Defatted coconut flour)

The serving styles of the formulated samples influenced the general perception and the ratings of the 15 panelists used for the sensory evaluation. The pictorial representations of the effect of the various serving styles on each of the attributes are shown in Figures 9-15. The charts revealed that the sample with 90:10 (AYB+Maize: Defatted coconut fiber) formulation was rated highest in terms of appearance when served with hot milk. This may be due to complete homogenization of the sample and milk by hot water, thereby presenting a more uniform appearance.

The consistency perception revealed that the control was most preferred when served with both cold and hot milk. This may not be unconnected with the fact that the judges would have been familiar with the control sample served with milk, since it is a commercially available product.

The judges also gave low scores for flavour, taste, after taste and overall acceptability to the control sample, especially when served with cold milk. The hot served samples got gelatinized and became very thick and they eventually got lower ratings for these parameters. It is important to note however, that almost all the perception ratings for all the serving styles were above average.

RATINGS

E E

RIBUT ATT

SAMPLE FORMULATION

Figure 9: Effect of Serving Style on the Appearance Perception of breakfast cereals made from blends of AYB + Maize: Defatted coconut flour

ATTRIBUTE RATINGS ATTRIBUTE

SAMPLE FORMULATION Figure 10: Effect of Serving Style on the Consistency Perception of breakfast cereals made from blends of AYB + Maize: Defatted coconut flour

Legend: A= 100:0 B=90:10 C=80:20 D=70:30 E=60:40 F=50:50 G= control (Weetabix) Sample ratio: AYB+ Maize: Defatted coconut fiber 67

RATINGS

E E

RIBUT ATT

SAMPLE FORMULATION Figure 11: Effect of Serving Style on the Flavour Perception of breakfast cereals made from blends of AYB + Maize: Defatted coconut flour

TTRIBUTE RATINGS TTRIBUTE A

SAMPLE FORMULATION

Figure 12: Effect of Serving Style on the Taste Perception of breakfast cereals made from blends of AYB + Maize: Defatted coconut flour

Lgend: A= 100:0 B=90:10 C=80:20 D=70:30 E=60:40 F=50:50 G= control (Weetabix) Sample ratio: AYB+ Maize: Defatted coconut fiber

RATINGS

ATTRIBUTE

SAMPLE FPRMULATION Figure 13: Effect of Serving Style on the Aftertaste Perception of breakfast cereals made from blends of AYB + Maize: Defatted coconut flour

E RATINGS E

ATTRIBUT

SAMPLE FORMULLATION

Figure 14: Effect of Serving Style on the Aftertaste Perception of breakfast cereals made from blends of AYB + Maize: Defatted coconut flour

Legend: A= 100:0 B=90:10 C=80:20 D=70:30 E=60:40 F=50:50 G= control (Weetabix) Sample ratio: AYB+ Maize: Defatted coconut fiber

ATTRIBUTE RATINGS ATTRIBUTE

SAMPLE FORMULATION

Figure 15: Effect of Serving Style on the Overall acceptability Perception of breakfast Cereals made from Blends of AYB + Maize: Defatted coconut flour

Legend: A= 100:0 B=90:10 C=80:20 D=70:30 E=60:40 F=50:50 G= control (Weetabix) Sample ratio: AYB+ Maize: Defatted coconut fiber

4.4.0 MINERAL COMPOSITION OF THE BREAKFAST CEREALS The mineral composition of the formulated breakfast cereals is shown in Table 14. These values are presented alongside the corresponding values obtained from the control sample (Weetabix) as well as the United States Recommended Dietary Allowance (USRDA) for each mineral value. Generally, significant differences (p<0.05) existed between the samples in almost all the parameters. The minerals decreased with increasing addition of defatted coconut flour in the formulations.

4.4.1 CALCIUM The Calcium content obtained from the samples indicated values ranging between 169±0.01mg/100g and 213±0.02mg/100g. The highest value occurred in the sample containing 100:0 formulation, while the least value occurred in the sample with 50:50 formulation. These values were higher than that recorded for the control sample- Weetabix (100mg/100g) and less than the US RDA (1000mg). Thus, 100g of the formulated samples can provide about 16.9- 21.3% of the US RDA. Lower values were also recorded for breakfast cereals made from maize, sorghum, soybeans and AYB composite flour (156±13.2mg/kg) (Agunbiade and Ojezele, 2010) and breakfast cereals made from sorghum and pigeon pea (137.05-156.34mg) (Mbaeyi, 2005).

Calcium is by far the most important mineral that the body requires and its deficiency is more prevalent than any other mineral (Kanu et al., 2009). Calcium, Phosphorus and vitamin D combine together to eliminate rickets in children and osteomalacia (the adult rickets) as well as osteoporosis (bone thinning) among older people (Adeyeye and Agesin, 2007). Since the products contain significant amounts of the element they can make an ideal meal for children and adults alike.

4.4.2 MAGNESIUM The Magnesium content obtained for the sample ranged from 29.0±0.02mg/100g to 43.0±0.01mg/100g. The highest value was recorded for the sample containing 50:50 formulation. These values were lower than the values recorded for the magnesium content of the control (92.00mg) and the US RDA which was 350mg for men and 280mg for women. Magnesium is an activator of many enzyme systems and maintains the electrical potential in the nerves (Adeyeye and Agesin, 2007). It works with calcium to assist in muscle contraction, blood clotting, and the regulation of blood pressure and lung functions (Swaminathan, 2003). 71

4.4.3 POTASIUM The potassium content of the breakfast cereals ranged from 88.0±0.02 to 191.0±0.02mg/100g. The highest value occurred in the sample containing 100:0 formulation. This range was lower than the value recorded for the control (545mg) but higher than the US RDA for both men and women (3.5mg). Higher values (70.19±6.82mg/kg) were recorded for fortified breakfast cereals (Agunbiade and Ojezele, 2010), while similar values (107.0- 238.0mg/100g) were recorded from breakfast cereals made from sorghum and pigeon pea (Mbaeyi, 2005). Potassium is primarily an intercellular cation, in large part this cation is bound to protein and with sodium influences osmotic pressure and contributes to normal pH equilibrium (Adeyeye and Agesin, 2007).

4.4.4 MANGANESE The manganese content of the samples ranged from 5.92±0.02 to 7.99±0.16 mg/100g. No value was recorded for the control sample (Weetabix) but the US RDA records 2.5mg/100g. The higher tolerable upper intake was 11mg/100g. Manganese functions as an essential constituent for bone structure, for reproduction and for normal functioning of the nervous system; it is also a part of the enzyme system. Manganese is readily found in nuts, whole grains, leafy vegetables, and tea (Adeyeye and Agesin, 2007; Ryan, 2009).

4.4.5 IRON The iron content of the products ranged from 9.81±0.30 to 14.10mg/100g. The values obtained in this study are higher than the values recorded for the control (5.16mg/100g) but lies within the range of the US RDA (10-15mg/100g). Similar results have been recorded (13.46±1.74) for breakfast cereals made from maize, sorghum, soybeans and AYB composite (Agunbiade and Ojezele, 2010). When foods with iron are eaten, it is absorbed into proteins and helps these proteins take in, carry, and release oxygen throughout the body. An iron deficiency called iron-deficiency anemia is very common around the world, especially for women and children in developing nations. Symptoms of iron deficiency take years to develop and include fatigue, weakness, and shortness of breath (Ryan, 2009).

4.4.6 COPPER The copper content of the samples revealed values ranging from 0.58±0.003 to 0.86±0.03mg/100g. These values were more than that of the control (0.23mg/100g), but less than the US RDA which is 1.5-3.0mg/100g. Copper and iron are present in the enzyme 72 cytochrome oxidase involved in energy metabolism. Copper deficiency is of little concern since it is widely distributed in other types of food (Adeyeye and Agesin, 2007). Copper makes up approximately 0.9g of the body. It can be found in some enzymes that are crucial to oxygen reactions and the way iron is metabolized. It also colors hair and skin, and helps form the protective shield around nerve fibers (Ryan, 2009).

4.4.7 SODIUM Results show that the sodium content of the samples ranged from 7.62+0.03 to 28.36+1.33mg/100g. These were far less than the value recorded for the control- Weetabix (387mg/100g) and the USRDA (500mg/100g). Higher sodium values (97.5-187.3mg/100g) were also reported for fortified breakfast cereals (Mbaeyi, 2005). Sodium is normally consumed in the form of salt. It is essential in the regulation of water content and in the maintenance of osmotic pressure of the body fluid. It also aids in the transport of CO2 in the blood. However, sodium is one of the minerals whose intake is considered a factor in the etiology of hypertension, hence its low intake is encouraged (Okaka, 2010).

4.4.8 ZINC The zinc content of the formulated samples showed a range of 1.97+0.05 to 3.35+0.01mg/100g. These values were higher than that recorded for the control- Weetabix (1.72mg/100g) but lower than the US RDA (15mg/100g- for men, 12mg/100g- for women). Agunbiade and Ojezele (2010) recorded lower values for fortified breakfast cereals as 1.54+0.30mg/kg and 1.64+0.4mg/kg. Zinc is a component of every living cell and plays a role in hundreds of bodily functions, from assisting in enzyme reactions to blood clotting, and is essential to taste, vision, and wound healing (Ryan, 2009).

The decreased level observed in some of the minerals may be associated with the processing techniques. Vegetable protein-containing raw materials were lost during soaking, boiling and frying. In any situation body mineral is threatened, supplementation may be contemplated (Agunbiade and Ojezele, 2010). 73

Table 14: Mineral Content of Breakfast Cereals made from Blends of AYB+Maize: Defatted Coconut (mg/100g)

Sample Ca Mg K Mn Fe Cu Na Zn

100:0 213+0.22a 430+0.01a 191+0.02a 7.99+0.16a 14.01+0.06a 0.86+0.01a 9.97+0.04 a 3.35+0.01a

90:10 204+0.03b 420+0.01a 113+0.03b 7.89+0.95a 13.83+0.04a 0.73+1.28b 9.97+0.04a 3.11+0.07a

80:20 191+0.02c 390+0.02ab 109+0.02c 7.41+0.12b 13.49+0.17b 0.73+0.07b 9.02+0.96b 2.80+0.32b

70:30 184+0.02d 380+0.01ab 103+0.02d 6.92+1.05b 12.12+0.26c 0.70+0.02b 8.23+1.30c 2.60+0.12b

60:40 172+0.02e 310+0.01 c 95.0+0.02e 6.10+0.10c 10.01+0.56d 0.58+0.03c 8.01+0.03c 2.11+0.05c

50:50 169+0.01e 290+0.06c 88.0+0.06 f 5.92+0.02c 9.81+0.30d 0.58+1.28c 7.62+0.03cd 2.11+0.05c

Weetabix 100 92.0 545 - 5.16 0.23 387 1.72

US RDA 1000 280-350 3.5 2-5 10-15 1.5-3 500 12-15 Data represents mean + SD (n=3) Means differently superscripted along the vertical column are significantly (P<0.05) different Legend:

USRDA= United States Recommended Dietary Allowance Sample: AYB+ Maize: defatted coconut fiber 74

4.5.0 VITAMINS COMPOSITION OF THE BREAKFAST CEREALS The results of the vitamin content of the breakfast cereals are shown in Table 15. The values are tabulated alongside the corresponding values for the control sample- Weetabix and the United States Recommended Dietary Allowance (USRDA) values for vitamins intake. Significant differences (p<0.05) were observed between most of the samples in the vitamins evaluated. The vitamins decreased with increase in the addition of defatted coconut flour.

4.5.1. VITAMIN B1 (THIAMINE) The values obtained for the thiamin content of the products ranged from 0.09+0.01 to 0.31+0.01mg/100g. These values are lower than those stated for the control sample (1.08mg/100g) and the USRDA (1.5mg/100g). Thus 100g of the formulated samples can provide 6-20% of vitamin B1 of the US RDA for adults and 10-33.3% for children between the ages 4-10.

4.5.2 VITAMIN B2 (RIBOFLAVIN)

The values for the vitamin B2 content of the products ranged from 0.32+0.10 to 0.43+0.02mg/100g, and were lower than the recorded values for the control (1.08mg/100g) and the US RDA (1.70mg/100g). Thus, 100g of the formulated samples can provide about

18.82- 25.3% of the US RDA for vitamin B2. Like thiamin, B12 acts as a coenzyme in the breakdown of fats, proteins, carbohydrate, and other nutrients. It also helps fatty acid reduction and also necessary for catabolism of nutrients in the liver. Furthermore, it assists eye and skin maintenance (White and Merrill, 1988)

4.5.3 VITAMIN B6

The results for B6 content of the breakfast cereals showed values ranging from 0.13+0.01 to 0.26+0.01mg/100g. These were lower than the values recorded for both the control sample (0.46mg/100g) as well as the US RDA (2.00mg/100g). Thus the formulated samples can provide about 6.5-13% of the US RDA for vitamin B6. B6 acts as a coenzyme for approximately 100 essential chemical reactions. These include protein and glycogen metabolism, proper action of steroid hormones, pyruvate production, production of red blood cells and much more. It assists in many decarboxylation reactions (removal of carboxyl group) for the production of several compounds such as glutamate (major neurotransmitter of the central nervous system). It also is of great use to the immune system in that it helps hemoglobin production and increases the amount of O2 carried by it (Bender, 1992).

4.5.4 VITAMIN B12

The results for the vitamin B12 content of the products ranged from 0.74+0.02 to 1.01+1.07mg/100g. These values are higher than the US RDA value (6µg). It was discovered that the commercial control sample does not contain vitamin B12. Vitamin B12 plays a large part in the conversion of homocysteine to methionine, which helps protect the heart from disease and also essential for the function and maintenance of the central nervous system, and severe deficiency in pernicious anemia produces a neurological disease of posterolateral spinal cord degeneration (Herbert and Das, 1999). It helps nerve cells, red blood cells, and the manufacturing/repair of DNA. It is vital for processing carbohydrates, proteins and fats, which help make all of the blood cells in our bodies (Bender, 1992).

4.5.5 VITAMIN C The results obtained for vitamin C content of the formulated samples ranged from 1.70+0.02 to 2.65+0.02mg/100g. These values are lower than the US RDA for men, women and children (30-60mg/100g), but it was discovered that the control sample does not contain vitamin C. Cordain (1999) reported that cereals contain no vitamin C or vitamin B12, no vitamin A and, apart from yellow corn, no beta-carotene.

Table 15: Vitamins Content of Breakfast Cereals made from Blends of AYB + Maize: Defatted Coconut Flour (mg/100g)

Sample B1 B2 B6 B12 C 100:00 0.31+0.01a 0.43+0.02a 0.26+0.02a 1.00+0.07a 2.65+0.02a

90:10 0.30+0.02a 0.41+0.02a 0.21+0.02b 1.00+0.02a 2.49+0.13a

80:20 0.19+0.01b 0.41+0.02a 0.20+0.06b 0.95+0.03ab 2.13+0.01b

70:30 0.13+0.02c 0.39+0.43a 0.20+0.02b 0.90+0.03b 2.10+0.02b

60:40 0.12+0.02d 0.33+0.02b 0.14+0.01c 0.88+0.03b 1.87+0.04bc

50:50 0.09+0.01e 0.32+0.10b 0.13+0.02c 0.74+0.02c 1.70+0.02bc

Weetabix 1.08 1.08 0.46 0.00 0.00

US RDA 1.50 1.70 2.00 6mcg 60.00

Means differently superscripted along the vertical column are significantly (P<0.05) different Values are mean of triplicate readings +SD

Sample ratio: AYB+ Maize: defatted coconut

4.6 ANTI-NUTRITIONAL CONTENT The anti-nutritional contents of the products are shown in Table 15. There were significant differences (p<0.05) in the samples as the level of the inclusion of defatted coconut flour increased.

4.6.1 PHYTATE/PHYTIC ACID The result obtained for the phytate content of the products ranged from 0.38 to 1.25mg/100g. A gradual decrease of the phytate was observed with increase in the level of the defatted coconut flour. Many dietary fibers contain phytic acid which binds minerals in the digestive tract, which eventually expels the minerals from the body. Some of these minerals are essential for good health, including zinc, iron and calcium. Although health experts recommend increasing intake of dietary fiber, eating too much fiber containing phytic acid can cause mineral deficiencies (Wasserman, 2010). Unlike many fiber sources, coconut dietary fiber does not contain phytic acid and, therefore, does not remove minerals from the body. Not only does coconut fiber prevent the removal of minerals, it also increases mineral absorption. Coconut fiber slows down the rate of emptying food from the stomach. This allows food more time in the stomach to release minerals, leading to higher levels of minerals available for the body to absorb (Wasserman, 2010).

The highest value of phytate was found in the sample containing 100:0 formulation. Legume seeds are known to constitute 1-3% of phytate and are dependent on species, cultivars and germination (Sridhar and Seena, 2006). The presence of vitamin C however, counteracts the inhibitory effects of phytate for consumption (Siegenberg et al., 1991).

4.6.2 OXALATE The results obtained for the oxalate content of the products ranged from 0.076 to 0.302mg/100g. The highest value was observed in the sample with 50:50 formulation. The oxalate content was directly proportional to the addition of the defatted coconut flour.

4.6.3 HEMAGLUTTININ The results for the hemagluttinin content of the products ranged from 0.10 to 0.29mg/100g. The highest value was observed in the sample with 50:50 formulation. The hemagluttinin content was inversely proportional to the addition of defatted coconut flour. However, there were no significant differences (p>0.05) between samples containing 70:30, 60:40 and 50:50 formulations as well as between samples containing 90:10 and 80:20 formulations. The 78 sample with 100:0 formulation, however, was significantly different (p<0.05) from other samples in the hemagluttinin contents.

4.6.4 TANNIN The tannin contents of the products were significantly (p<0.05) low and ranged from 0.00064 to 0.0016mg/100g. Higher values (0.035 to 0.130mg/100g) were recorded for breakfast cereals made from Pigeon pea and Sorghum (Mbaeyi. 2005). Tannins are located in the seed coat of the grains and are known to have deleterious effects due to their strong interactions with proteins, with the resulting complexes which are not readily digested by monogastrics. This lowers the protein digestibility, PER and weight (Mbaeyi. 2005; El-Niely, 2007).

Although legumes contain a wide range of toxic components, the term toxic being referred to „an adverse physiological response produced in man or animals by a particular food or substance derived there from, the effects of most of these components are small or negligible in a mixed diet especially when legumes are properly cooked. During the processing of legumes it is important that toxic components be reduced to levels that pose no threat to health (Walker and Ochhar, 1982).

Table 16: Anti-Nutritional Factors of the Formulated Samples (mg/100g)

Sample phytate Oxalate Hemagluttinin Tannin (unit/mg)

100:0 1.25+0.01a 0.08+0.01d 0.29+0.01a 0.0016+0.0001a

90:10 1.13+0.01b 0.15+0.01c 0.18+0.01b 0.0013+0.0001b

80:20 1.00+0.1c 0.15+0.01c 0.17+0.01b 0.0013+0.0001b

70:30 1.00+0.1c 0.23+0.01b 0.11+0.01c 0.00084+0.00001c

60:40 0.50+0.01d 0.23+0.01b 0.10+0.01c 0.00075+0.00001cd

50:50 0.38+0.01e 0.30+0.01a 0.10+0.01c 0.00064+0.00001d

Values are means +SD of triplicate determinations Means differently superscripted along the vertical columns are significantly different (p<0.05) Sample ratio: AYB+ Maize: Defatted coconut fiber

4.7 AMINO ACID PROFILE OF THE BREAKFAST CEREALS Figure 16 shows the amino acid profile of the breakfast cereals. The results reveal that the products contained varying amounts of both essential and non-essential amino acids. Aspartic acid, glutamic acid and proline recorded the least values, while higher values were recorded for threonine, leucine and glycine. Apart from isoleucine which had similar values with the United States Recommended Dietary Allowance (USRDA) and valine, which had slightly lower values, the essential amino acids in all the products were higher than the USRDA values (Weetabix, 2010). It is important to note that the consumption of these products with milk will make up for all the required amino acids lacking in the products.

Although proteins from plant sources tend to have a relatively low biological value, in comparison to protein from eggs or milk, they are nevertheless "complete" in that they contain at least trace amounts of all of the amino acids that are essential in human nutrition. Eating various plant foods in combination can provide a protein of higher biological value (McDougall, 2002).

Figure 16: Amino Acid Profile of Breakfast Cereals Made From Blends of AYB + Maize: Defatted Coconut Flour (mg/100g)

Legend: A=100:0, B= 90:10, C=80:20, D=70:30, E=60:40, F=50:50 Sample ratio: AYB+ Maize: Defatted Coconut Flour

4.8 MICROBIAL EXAMINATION The microbial examination of the products revealed different values for total viable count, molds and coliform counts, as shown in Figure 17. The total viable count ranged from 0.5 to 1.51x102Cfu/g, while the mold count ranged between 0.00 to 6.0x10Cfu/g. The contamination could have occurred during cooling and before packaging.

Yeasts are commonly present as contaminants in cereals and can probably be attributed to the low value of the pH which creates ideal conditions for yeast growth (Serna-Saldivar and Rooney, 1995). The presence of micro flora may be also due to availability of more nutrients for microbial proliferation and enhanced metabolic activities (Mbata et al., 2009). However, the samples had low levels of bacteria and mold growth. No coliform was detected. Thus the consumption of these products may not be fraught by the danger of contacting any food borne disease.

Figure 17: Microbial Content of Freshly Prepared Breakfast Cereals made from Blends of AYB + Maize: Defatted Coconut flour Legend: A=100:0, B= 90:10, C=80:20, D=70:30, E=60:40, F=50:50 Sample ratio: AYB+ Maize: Defatted Coconut Flour

CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATIONS

5.1 CONCLUSION The study showed that acceptable ready-to-eat breakfast cereals could be produced from maize, African yam bean and defatted coconut flour. Evaluation of the products showed values that compared favourably with the commercial control sample (Weetabix) as they have been shown to be good sources of protein, energy, vitamins and minerals.

The study has shown that producing breakfast cereals with seed legumes could boost the protein level (up to 18%) in the final products. It also played a role in providing micronutrients like minerals and vitamins, especially Vitamin B12 and C which are absent in the commercial control sample. The roasting process employed in the study played a role in reducing the relatively high level of anti-nutrients associated with leguminous food sources. The process also influenced low moisture content (3-4%) of the products, which is important for transportation and extension of the shelf life of properly packaged products. Furthermore, it limited the micro-flora of the final products to insignificant levels; thereby making the products safe for consumption. The introduction of defatted coconut fiber increased the fiber content of the final products, although it gradually reduced the overall nutritional value. It was however aimed at increasing bulk and aiding digestion. Most of the formulated samples were scored above average by sensory judges and showed some similarities with Weetabix (control) (p>0.05), implying its potential acceptability when commercialized.

The seeds of African yam beans are projected by the findings of this work to be promising cheap source of nutrients that are lacking in most expensive ready-to-eat food products and could also play a key role in the acceptability and nutritional value of monotonous diets in the world at large.

5.2 RECOMMENDATIONS More studies should be carried out on the products to determine their health benefits on humans such as insulin sensitivity, as AYB has been proposed in previous research findings as beneficial to diabetics and patient with other relative illnesses.

Further research should be carried out to ascertain the shelf life and the best packaging recommended for the formulated samples. These, along with other factors will influence the commercialization of the products for national sustenance.

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Adewale, D and Dominique, D. (2009). African yam bean: A Crop with Food Security Potentials for Africa. Genetic Resources Centre, IITA, Ibadan.

Agunbiade, S.O. and Longe, O.G. (1998). African yam bean hull Chemical Composition and its effects on Rat‟s Mineral Retention, Serum Biochemical Components and Enzymatic Activities. Hahrung, 42 (2): 89-93.

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APPENDIX I

SENSORY EVALUATION SCORE SHEET

Instructions

1) You are served coded samples of instant breakfast cereals 2) You are requested to take a sip of water to gaggle your mouth before tasting each sample. 3) Rate the samples according to your degree of acceptance from 1-9 as shown below. 4) Enter the appropriate scale in the box provided for each attribute.

Attributes A B C D E F G Colour Consistency Flavour Taste Aftertaste Mouth feel Overall acceptability

Extremely like = 9, Very much like = 8, Moderately like = 7, Slightly like = 6, Neither like nor dislike = 5, Slightly dislike = 4, Moderately dislike = 3, Very much dislike = 2, Extremely dislike = 1.

Which sample do you like most? ……………………………………………………………………………………………….

Reason(s) for preference:……………………………………………………………

………………………………………………………………………………………

Any other comments:

APPENDIX II

AMINO ACID PROFILE OF FORMULATED BREAKFAST CEREALS (mg/100g)

PhenyLamine Valine throsine Tryptophane Isoleucine Methionine Histidine Argenine Lysine Leucine Crysteine Alani Tyrosine Glycine Serine A Aspetic. Glutamic. A. Asparagine Glutamine Proline

SAMPLE

100:0 320 240 810 520 220 100 240 510 250 810 340 220 560 750 120 40 40 520 310 50

90:10 310 210 730 500 190 100 210 470 240 770 310 190 490 720 110 40 30 480 300 50

80:20 290 180 730 420 180 90 210 390 210 680 280 180 440 550 100 30 30 250 290 50

70:30 250 180 710 410 150 90 190 380 170 650 250 150 390 530 80 20 20 220 280 40

60:40 220 160 660 400 180 80 180 210 130 610 210 130 310 520 70 20 10 210 160 40

50:50 190 200 560 380 150 70 160 180 90 590 109 110 280 460 50 10 10 190 140 30

Sample: AYB+ Maize : defatted coconut fiber

APPENDIX III

RAW VALUES FOR MICROBIAL PROFILE OF BREAKFAST CEREALS MADE FROM BLENDS OF AYB+MAIZE: DEFATTED COCONUT FLOUR

Samples Bacteria Count, Mould Count, Coliform Count, Cfu/g Cfu/g Cfu/g

100:0 0.5x10 0.0x10 0.0x10

90:10 0.8x10 0.1x10 0.0x10

80:20 1.1x10 0.2x10 0.0x10

70:30 1.6x10 0.25x10 0.0x10

60:40 2.4x10 0.3x10 0.0x10

50:50 1.51x102 0.6x10 0.0x10

Means of Cfu/g as indices of microbial stability of samples Sample: AYB+maize : defatted coconut fiber

APPENDIX IV ANOVA TABLE FOR ANTI-NUTRIENTS STATISTICAL ANALYSIS

Sum of Squares df Mean Square F Sig.

PHYTATE Between Groups 1.868 5 .374 109.859 .000

Within Groups .041 12 .003

Total 1.908 17

OXALATE Between Groups .092 5 .018 183.600 .000

Within Groups .001 12 .000

Total .093 17

HEMAGLUTTINNIN Between Groups .078 5 .016 156.000 .000

Within Groups .001 12 .000

Total .079 17

TANNIN Between Groups .000 5 .000 86.362 .000

Within Groups .000 12 .000

Total .000 17

APPENDIX V ANOVA TABLE FOR SENSORY DATA OF FORMULATED BREAKFAST CEREALS SERVED RAW

Sum of Squares df Mean Square F Sig.

COLOUR Between Groups 13.581 6 2.263 1.102 .367

Within Groups 201.333 98 2.054

Total 214.914 104

CONSISTENCY Between Groups 24.895 6 4.149 2.652 .020

Within Groups 153.333 98 1.565

Total 178.229 104

FLAVOUR Between Groups 25.429 6 4.238 2.562 .024

Within Groups 162.133 98 1.654

Total 187.562 104

TASTE Between Groups 27.790 6 4.632 2.741 .017

Within Groups 165.600 98 1.690

Total 193.390 104

AFTER TASTE Between Groups 33.295 6 5.549 3.113 .008

Within Groups 174.667 98 1.782

Total 207.962 104

MOUTH FEEL Between Groups 12.724 6 2.121 1.469 .197

Within Groups 141.467 98 1.444

Total 154.190 104

OVERALL ACC. Between Groups 25.714 6 4.286 2.933 .011

Within Groups 143.200 98 1.461

Total 168.914 104

APPENDIX VI

ANOVA TABLE FOR SENSORY DATA OF FORMULATED BREAKFAST CEREALS SERVED WITH COLD WATER

Sum of Squares df Mean Square F Sig.

Colour Between Groups 59.176 6 9.863 2.263 .043

Within Groups 427.014 98 4.357

Total 486.190 104

Consistency Between Groups 36.650 6 6.108 1.203 .311

Within Groups 497.579 98 5.077

Total 534.229 104

Flavour Between Groups 22.267 6 3.711 .843 .540

Within Groups 431.295 98 4.401

Total 453.562 104

Taste Between Groups 39.672 6 6.612 1.469 .197

Within Groups 441.185 98 4.502

Total 480.857 104

Aftertaste Between Groups 27.569 6 4.595 .906 .494

Within Groups 497.231 98 5.074

Total 524.800 104

Mouthfeel Between Groups 48.281 6 8.047 1.673 .136

Within Groups 471.281 98 4.809

Total 519.562 104 overall acceptability Between Groups 13.134 6 2.189 .403 .875

Within Groups 531.666 98 5.425

Total 544.800 104

100

APPENDIX VII

ANOVA TABLE FOR SENSORY DATA OF FORMULATED BREAKFAST CEREALS SERVED WITH COLD MILK

Sum of Squares df Mean Square F Sig.

Colour Between Groups 6.057 6 1.010 .645 .694

Within Groups 153.333 98 1.565

Total 159.390 104

Consistency Between Groups 47.695 6 7.949 7.312 .000

Within Groups 106.533 98 1.087

Total 154.229 104

Flavour Between Groups 39.657 6 6.610 5.071 .000

Within Groups 127.733 98 1.303

Total 167.390 104

Taste Between Groups 51.581 6 8.597 6.047 .000

Within Groups 139.333 98 1.422

Total 190.914 104

Aftertaste Between Groups 40.800 6 6.800 4.636 .000

Within Groups 143.733 98 1.467

Total 184.533 104

Mouthfeel Between Groups 69.733 6 11.622 8.655 .000

Within Groups 131.600 98 1.343

Total 201.333 104

Overall acceptability Between Groups 57.562 6 9.594 8.031 .000

Within Groups 117.067 98 1.195

Total 174.629 104

101

APPENDIX VIII

ANOVA FOR SENSORY DATA OF FORMULATED BREAKFAST CEREALS SERVED WITH HOT MILK

Sum of Squares df Mean Square F Sig.

Colour Between Groups 11.562 6 1.927 1.250 .288

Within Groups 151.067 98 1.541

Total 162.629 104

Consistency Between Groups 49.657 6 8.276 6.583 .000

Within Groups 123.200 98 1.257

Total 172.857 104

Flavour Between Groups 42.648 6 7.108 4.379 .001

Within Groups 159.067 98 1.623

Total 201.714 104

Taste Between Groups 35.657 6 5.943 3.102 .008

Within Groups 187.733 98 1.916

Total 223.390 104

Aftertaste Between Groups 41.448 6 6.908 3.511 .003

Within Groups 192.800 98 1.967

Total 234.248 104

Mouthfeel Between Groups 54.857 6 9.143 5.490 .000

Within Groups 163.200 98 1.665

Total 218.057 104

Overall acceptability Between Groups 33.790 6 5.632 3.578 .003

Within Groups 154.267 98 1.574

Total 188.057 104

102

APPENDIX IX

ANOVA TABLE FOR FUNCTIONAL PROPERTIES ANALYSIS

Sum of Squares Df Mean Square F Sig.

Ph Between 9.180 5 1.836 18359.300 .000 Groups

Within Groups .001 12 .000

Total 9.181 17

Bulk density Between .034 5 .007 54.918 .000 Groups

Within Groups .001 12 .000

Total .035 17

Water abs cap Between 158.194 5 31.639 66220.709 .000 Groups

Within Groups .006 12 .000

Total 158.200 17

Oil abs cap Between .404 5 .081 807.200 .000 Groups

Within Groups .001 12 .000

Total .405 17

Foam cap Between 2.812 5 .562 6326.763 .000 Groups

Within Groups .001 12 .000

Total 2.813 17

Viscosity Between 314.189 5 62.838 628378.400 .000 Groups

Within Groups .001 12 .000

Total 314.190 17

In-vitro PD Between 494.963 5 98.993 128191.799 .000 Groups

Within Groups .009 12 .001

Total 494.972 17

Gelation cap Between 412.669 5 82.534 825337.200 .000 Groups

Within Groups .001 12 .000

Total 412.670 17 103

APPENDIX X

ANOVA TABLE FOR PROXIMATE COMPOSITION ANALYSIS

Sum of Squares df Mean Square F Sig.

N% Between Groups .323 5 .065 363.281 .000

Within Groups .002 12 .000

Total .325 17

PROTEIN% Between Groups 12.624 5 2.525 383.196 .000

Within Groups .079 12 .007

Total 12.703 17

FAT% Between Groups .252 5 .050 206.441 .000

Within Groups .003 12 .000

Total .255 17

ASH% Between Groups 9.386 5 1.877 3519.90 .000 0

Within Groups .006 12 .001

Total 9.393 17

CRUDEFIBER% Between Groups 11.847 5 2.369 4.376 .017

Within Groups 6.497 12 .541

Total 18.344 17

MOISTURE% Between Groups 1.487 5 .297 225.898 .000

Within Groups .016 12 .001

Total 1.503 17

CARBOHYDRATE% Between Groups 12.547 5 2.509 4.249 .019

Within Groups 7.087 12 .591

Total 19.634 17

104

APPENDIX XI

ANOVA TABLE FOR VITAMIN ANALYSIS

Sum of Squares df Mean Square F Sig. vitB1(ppm) Between Groups 12.332 5 2.466 11095.654 .000

Within Groups .003 12 .000

Total 12.334 17 vitB2(ppm) Between Groups 3.083 5 .617 584.140 .000

Within Groups .013 12 .001

Total 3.096 17 vitB12(ppm) Between Groups 14.768 5 2.954 2671.582 .000

Within Groups .013 12 .001

Total 14.781 17 vitC(ppm) Between Groups 194.012 5 38.802 11918.834 .000

Within Groups .039 12 .003

Total 194.051 17 vitB6(ppm) Between Groups 3.802 5 .760 1160.042 .000

Within Groups .008 12 .001

Total 3.810 17

105

APPENDIX XII

RDA OF VITAMINS FOR CHILDREN AND ADULTS (mg/kg of body weight)

Age Ascorbic Folacin/ Niacin Riboflavin Thiamine Vitamin Vitamin B12 Acid Folate B6

mg Mcg Mg Mg mg Mg Mcg

Children 4-6 40/45 200/75 12 1.1 0.9 0.9/1.1 1.5/1.0

7-10 40/45 300/100 16/13 1.2 1.2/1.0 1.2 2.0/1.4

Males 15-18 45/60 400/200 20 1.8 1.5 2.0 3.0/2.0

19-24 45/60 400/200 20/19 1.8/1.7 1.5 2.0 3.0/2.0

25-50 45/60 400/200 18/19 1.6/1.7 1.4/1.5 2.0 3.0/2.0

50+ 45/60 400/200 16/15 1.5/1.4 1.2 2.0 3.0/2.0

Females 15-18 45/60 400/180 14/15 1.4/1.3 1.1 2.0/1.5 3.0/2.0

19-24 45/60 400/180 14/15 1.4/1.3 1.1 2.0/1.6 3.0/2.0

25-50 45/60 400/180 13/15 1.2/1.3 1.0/1.1 2.0/1.6 3.0/2.0

50+ 45/60 400/180 12/13 1.1/1.2 1.0 2.0/1.6 3.0/2.0 * First figure refers to the old RDA listing while the second figure refers to the newer DRI listing (www.nap.edu) 106

APPENDIX XIII

DATA FOR MINERAL REQUIREMENTS FOR CHILDREN AND ADULTS

Age Calcium Phosphorous Iodine Iron Magnesium Zinc Selenium Fluoride

mg Mg ug mg mg mg *ug *mg

Children 4-6 800 800/500 80/90 10 200/130 10 -/20 -/1.1

7-10 800 800 110/120 10 250 10 -/30 -/3.2

Males 15-18 1200/1300 1200/1250 150 18/12 400/410 15 -/50 -/3.8

19-24 800/1000 800/700 140/150 10 350/400 15 -/70 -/3.8

25-50 800/1000 800/700 130/150 10 350/420 15 -/70 -/3.8

50+ 800/1200 800/700 110/150 10 350/420 15 -/70 -/2.9

Females 15-18 1200/1300 1200/1250 115/150 18/15 300/360 15/12 -/50 -/3.1

19-24 800/1000 800/700 100/150 18/15 300/310 15/12 -/55 -/3.1

25-50 800/1000 800/700 100/150 18/15 300/320 15/12 -/55 -/3.1

50+ 800/1200 800/700 80/150 10 300/320 15/12 -/55 -/3.1 * first figure refers to the old RDA listing while the second figure refers to the newer DRI listing -. www.nap.edu

107

APPENDIX XIV

RDA OF ESSENTIAL AMINO ACIDS FOR CHILDREN AND ADULTS

Requirement - mg. per kg. of body weight Infant Child Adults Amino acid 3 - 6 mo. 10 - 12 yr.

Histidine 33 not known not known

Isoleucine 80 28 12

Leucine 128 42 16

Lysine 97 44 12 S-containing amino 45 22 10 acids Aromatic amino acids 132 22 16

Threonine 63 28 8

Tryptophan 19 4 3

Valine 89 25 14 www.nap.edu