University of Ghana http://ugspace.ug.edu.gh CONSUMPTION PATTERNS, PERCEPTIONS AND TOTAL CAROTENOIDS, IRON AND ZINC CONTENTS OF YELLOW FLESH CASSAVA. THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA BY ELIZABETH AFRIYIE DUAH (10311317) IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MASTERS OF PHILOSOPHY (MPHIL) NUTRITION DEGREE JULY, 2016 University of Ghana http://ugspace.ug.edu.gh DECLARATION I, Elizabeth Afriyie Duah hereby declare that this study was solely carried out by me under the supervision of Prof. Matilda Steiner- Aseidu and Prof. Angelina O. Danquah in the Department of Nutrition and Food Science , University of Ghana, Legon and that this thesis either whole or part has not been presented for another degree elsewhere. ………………………………………. ………………………….. Elizabeth Afriyie Duah (Date) (Student) ………………………… ………………………… Prof. Matilda Steiner –Aseidu (Date) (Supervisor) .................. .…………. ………………………… Prof. Angelina O. Danquah (Date) (Co- Supervisor) i University of Ghana http://ugspace.ug.edu.gh DEDICATION To the Almighty God and the Holy Spirit who has been my source of my wisdom, inspiration, focus and strength throughout my study. I dedicate this great piece of work to Prof. F.K Saalia who has been a wonderful teacher and a great source of motivation for this research. And to my family, who have been supportive in every way from the beginning to the end of this degree. ii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS All glory to the Almighty God who started this work and ended it with me. I am forever grateful to Him. I would like to thank the Department of Nutrition and Food Science for the opportunity given me to study for this MPhil Degree. My grateful thoughts turn to Prof. F.K Saalia and to my supervisors Prof. M.Steiner- Aseidu and Prof A.O. Danquah for their keen interest and patience in developing and guiding the research work, and for reading every aspect of the thesis and making constructive criticisms. I say with utmost sincerity, thank you and may the good Lord continue to use you to achieve more. I would also like to thank International Institute of Tropical Agriculture (IITA) for funding this research and particularly Rev. Dr. Elizabeth Parkes. I would also like to thank HarvestPlus and Crops Research Institute for their support in making this research a success. I would like to express special thanks to Dr. Godfred Egbi of Noguchi Memorial Institute, Mr. Anthony Acquatey–Mensah, Mr. Humphrey Thompson, and Miss Senam Klomegah for their various supportive roles in ensuring the success of the work. To all the laboratory technicians in the Nutrition and Food Science Department, I would also like to show gratitude for their support. I am greatly indebted to my family for their prayers and their support during my period of studies. To all others too numerous to count I would like to say thank you and God bless you. iii University of Ghana http://ugspace.ug.edu.gh Table of Contents DECLARATION............................................................................................................................ i DEDICATION............................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................ iii ABSTRACT .................................................................................................................................. xi CHAPTER 1 .................................................................................................................................. 1 1.0 INTRODUCTION ............................................................................................................... 1 1.1 Background information ................................................................................................. 1 1.2 Problem statement ....................................................................................................... 3 1.3 Research questions .............................................................................................................. 4 1.4 Main objective of study ....................................................................................................... 4 1.5 Specific objectives ................................................................................................................ 4 2.0 LITERATURE REVIEW ................................................................................................... 6 2.1 The role of cassava in nutrition and health ................................................................... 6 2.1.1 Nutritional problems associated with cassava consumption .................................. 6 2.1.2 Effects of processing on the nutritional value of cassava ....................................... 7 2.1.3 Cassava utilization and consumption in Ghana......................................................... 8 2.2 The role of vitamin A and carotenoids in human health and nutrition ..................... 8 2.2.1 Dietary recommendations for vitamin A and carotenoids.................................... 10 2.3 Role of antioxidants in human nutrition ..................................................................... 10 2.4 Effects of processing on carotenoids ............................................................................ 11 2.5 Bio-availability of carotenoids .................................................................................... 12 2.5.1 Factors that affect bioavailability of carotenoids .................................................... 12 2.5.2 Estimation of bio-availability of pro-vitamin A. .................................................... 14 2.6 Methods for determination of total carotenoids ......................................................... 14 2.7 Methods of beta carotene analysis ............................................................................. 15 2.8 Iron and zinc in nutrition and health......................................................................... 15 2.8.1 The interrelation of vitamin A, iron and zinc in human metabolism .................. 17 2.9 Micronutrient deficiencies in West Africa .................................................................. 17 2.9.1 Magnitude and consequences of vitamin A deficiency in Ghana ......................... 18 2.9.2 Intervention strategies to combat vitamin A deficiency........................................ 19 iv University of Ghana http://ugspace.ug.edu.gh 2.9.5 Bio-fortification ......................................................................................................... 21 2.9.5 Bio-fortification of crops in Africa. ......................................................................... 22 2.9.6 Bio-fortification of cassava ...................................................................................... 23 2.9.7 The nutritional value of yellow flesh cassava ......................................................... 23 2.9.8 Effects of processing on the carotenoid retention of yellow flesh cassava. .......... 24 2.9.9 Bio-availability of carotenoids in yellow flesh cassava .......................................... 25 2.9.10 Consumer acceptability, perceptions and knowledge on bio-fortified crops .... 26 CHAPTER 3 ................................................................................................................................ 28 3.0 METHODOLOGY ............................................................................................................ 28 3.1 Laboratory study ............................................................................................................. 28 3.1.1 Sample collection and preparation ............................................................................... 28 3.1.2 Preparation of gari...................................................................................................... 29 3.1.3 Preparation of kokonte ............................................................................................... 29 3.1.4 Preparation of boiled roots and leaves ..................................................................... 29 3.1.5 Determination of moisture content ........................................................................... 29 3.1.6 Quantification of Beta carotene in yellow cassava genotypes ................................. 30 3.1.7 Determination of total carotenoids in fresh yellow cassava roots .......................... 30 3.1.7.4 Yellow cassava gari ............................................................................................... 32 3.1.8 Determination of iron and zinc contents .................................................................. 33 3.1.10 Determination of carotenoid retention after processing ....................................... 34 3.1.11 Determination of in vitro bio-accessibility of carotenoids in yellow flesh cassava .... 34 3.2 Cross sectional study ......................................................................................................... 36 3.2.1 Study area and population ............................................................................................ 36 3.2.2 Sample size determination and sampling technique for the survey ....................... 36 3.3 Data analyses ..................................................................................................................... 37 3.3.1 Laboratory data ........................................................................................................ 37 3.3.2 Survey data .................................................................................................................. 37 3.4 Ethical Consideration .................................................................................................... 38 4.0 RESULTS ......................................................................................................................... 39 4.1 Preamble ............................................................................................................................ 39 4.2 Laboratory analysis........................................................................................................... 40 v University of Ghana http://ugspace.ug.edu.gh 4.2.1 Total carotenoids, iron and zinc contents of fresh yellow cassava roots ............... 40 4.2.2 Total carotenoids, iron and zinc contents of fresh yellow cassava leaves ................... 41 4.2.4 Total carotenoids, iron and zinc contents of yellow flesh cassava products ................. 44 4.2.4.1 Total carotenoids, iron and zinc contents of yellow flesh gari .................................... 44 4.2.4 .2 Nutrient profile of yellow flesh kokonte ..................................................................... 45 4.2.4.3 Total carotenoids, iron and zinc contents of boiled yellow flesh cassava roots ......... 45 4.2.4.4 Total carotenoids, iron and zinc contents of boiled yellow flesh cassava leaves ..... 47 4.2.5 β -carotene contents in selected yellow cassava roots and leaves and their products .. 48 4.2.6 Total antioxidant activity of yellow flesh cassava products ........................................... 49 4.30 Total carotenoid retention in yellow cassava products after processing ....................... 50 4.3.1 Effect of processing and variety on total carotenoids, iron and zinc contents of yellow flesh cassava ................................................................................................................................. 51 4.3.2 Contribution of yellow flesh cassava to RDA of vitamin A for under-5 children. ... 54 4.3.4 In vitro bio-accessibility of carotenoids in yellow flesh cassava................................. 56 4.3.5.1 Socio-demographic characteristics of study population ...................................... 58 4.3.5.2 Consumption patterns of white cassava ................................................................ 60 4.3.5.3 Reasons for cassava consumption among Ghanaians ........................................ 61 4.3.5.4 Knowledge of nutritive value of white and yellow flesh cassava ......................... 61 4.3.5.5 Acceptability of yellow flesh cassava ..................................................................... 62 4.3.5.6 Perceived usage of yellow flesh cassava ............................................................... 63 CHAPTER 5 ................................................................................................................................ 66 5.0 DISCUSSION .................................................................................................................... 66 5.1 Nutrient profiles of yellow flesh cassava roots and leaves ............................................. 66 5.2 Effect of processing on total carotenoids, iron and zinc contents of yellow cassava ... 68 5.3 Factors affecting carotenoid retention .......................................................................... 70 5.4 In vitro bio-accessibility of carotenoids in yellow cassava............................................. 71 5.5 Contribution of yellow flesh cassava to RDA of vitamin A under- 5 children ............ 71 5.6 Effect of processing and cultivar on antioxidant activity of carotenoids ..................... 71 5.7 Comparison of icheck and spectrophotometric method for carotenoid determination ................................................................................................................................................... 72 5.8 Socio-demographics characteristics................................................................................. 72 vi University of Ghana http://ugspace.ug.edu.gh 5.9 Consumption patterns of white cassava .......................................................................... 73 5.10 Acceptability of yellow flesh cassava ....................................................................... 73 5.11 Determinants of knowledge and willingness to accept yellow flesh cassava .......... 74 5.12 Limitations of the study ................................................................................................ 74 CHAPTER 6 ................................................................................................................................ 75 6.0 CONCLUSION AND RECOMMENDATIONS .......................................................... 75 6.1 Conclusions ........................................................................................................................ 75 6.2 Recommendations ............................................................................................................. 76 REFERENCES ............................................................................................................................ 77 APPENDICES ........................................................................................................................... 96 APPENDIX I ............................................................................................................................. 96 APPENDIX II ........................................................................................................................... 97 APPENDIX IV .......................................................................................................................... 99 APPENDIX V ......................................................................................................................... 102 APPENDIX VI ........................................................................................................................ 103 vii University of Ghana http://ugspace.ug.edu.gh LIST OF ACRONYMS AND ABBREVIATIONS IITA International Institute of Tropical Agriculture AOAC Association of Analytical Communities CSRI Council for Scientific Research Institute DPPH 1,1-Diphenyl-2-Picryl-Hydrazyl CRI Crops Research Institute EAR Estimated Average Requirements FAO Food and Agriculture Organization FAOSTAT Food and Agriculture Organization Statistics HPLC High Performance Liquid Chromatography IU International Units MICS Multiple Indicator Cluster Survey MOST Micronutrient Program RDA Recommended Daily Allowance T.C Total Carotenoids TAG Triacylglycerol USAID United States Agency for International Development WHO World Health Organization viii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1: Total carotenoids, iron and zinc contents of fresh yellow flesh cassava roots (d.m.b) .. 40 Table 2: Total carotenoids, iron and zinc contents of fresh yellow cassava leaves (d.m.b) ..... 41 Table 9: Total antioxidant activity (%) of yellow flesh cassava products .................................... 50 Table 10: Total antioxidant activity in yellow cassava products in alpha -tocopherol units ...... 50 Table 11: Total carotenoid retention (%) in yellow cassava products after processing ............... 51 Table 12: Statistical effect of processing on total carotenoids, iron and zinc contents of yellow flesh cassava.................................................................................................................................. 52 Table 13: Statistical effect of cultivar on total carotenoids, iron and zinc contents of yellow flesh cassava .......................................................................................................................................... 52 Table 14: Statistical effects of processing on antioxidant activity of yellow flesh cassava ......... 53 Table 15: Statistical effect of cultivar on antioxidant activity of yellow flesh cassava .............. 53 Table 16: In -vitro bio-accessibility (%) of carotenoids in yellow flesh cassava ......................... 56 Table 17: Effect of processing on the in vitro bio accessibility of carotenoids ............................ 57 Table 18: Effect of cultivar on the in vitro bio-accessibility of carotenoids ................................ 57 Table 19: Socio-demographic characteristics of study population (N=287) ................................ 59 Table 20: Predictors of Knowledge and „willingness to accept‟ Yellow Flesh Cassava (N=284) 65 ix University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 1: Contribution of yellow flesh cassava to recommended daily allowance (RDA) of vitamin A ...................................................................................................................................... 54 TM Figure 2: Relationship between iCheck carotene and spectrophotometric method (Gari) ....... 55 TM Figure 3: Relationship between iCheck carotene and spectrophotometric method (Fresh roots) ....................................................................................................................................................... 56 Figure 4: Consumption frequency of cassava products ................................................................ 60 Figure 5: Reasons for cassava consumption ................................................................................. 61 Figure 6: Knowledge of yellow flesh cassava .............................................................................. 62 Figure 7: Acceptability of yellow flesh cassava ........................................................................... 63 Figure 8: Perceived usage of yellow flesh .................................................................................... 64 x University of Ghana http://ugspace.ug.edu.gh ABSTRACT Background: In Ghana, more than 60 % of under 6 year old children are estimated to suffer from sub-clinical vitamin A deficiency. Bio-fortification is the process of breeding nutrients into crops and it provides a relatively economical, sustainable way of giving out more micronutrients. Cassava is also the leading crop in terms of energy intake and per capita consumption in Ghana thus bio-fortifying cassava with provitamin A can improve the vitamin A composition of this staple food and provide a relatively economical, sustainable way of giving out more vitamin A to the poor. This will reduce the prevalence of severely malnourished people who require treatment by interventions with supplementary foods, as well as help them maintain improved nutritional status. Yellow flesh cassava proposes to be an avenue to solving micronutrient deficiencies specifically vitamin A in Africa. There is therefore the need to determine the nutrient profile, perception and knowledge of yellow flesh cassava. Objective: The main objective was to investigate consumption patterns of white cassava, knowledge, perceptions and the nutritive profiles of yellow flesh cassava roots and leaves and its relationship to the recommended daily allowance for vitamin A. Methods: Total carotenoids, iron and zinc contents in fresh yellow cassava leaves, roots and products (gari, konkonte, boiled cassava roots and leaves), antioxidant activity and the in vitro bio accessibility of the carotenoids were determined using standard methods. A cross sectional survey was carried out to identify the consumption patterns of white cassava, the knowledge and perception of yellow flesh cassava among Ghanaians. xi University of Ghana http://ugspace.ug.edu.gh Results: Total carotenoids (T.C), iron (Fe) and zinc (Zn) for fresh roots ranged from 4.73±0.11 to 10.11±0.18 µg/g; 87.35±3.18 to 146.25±1.20 mg/100g; 0.30± 0.01 to1.55±0.07 mg/100g, respectively. T.C, Fe and Zn for fresh leaves ranged from 792.93±0.98 to 2649.20±29.10 µg/g; 118.35±0.07 to 182.05±0.07 mg/100g; 3.75±0.64 to 15.50±0.14 mg/100g respectively. T.C for gari was between 3.21±2.79 and 7.39±1.06 µg/g, iron 118.75±0.64 to 181.85±2.05 mg/100g and zinc, 0.25±0.07 to 0.80±0.14 mg/100g. Iron and zinc contents for the kokonte samples ranged from 101.45±0.64 to 116.30± 0.14 mg/100g and 0.15±0.07 to 0.70±0.01 mg/100g, however T.C was not detectable. T.C for boiled roots were between 1.22±0.05 and 2.14±0.11µg/g. Iron content for the samples ranged from 118.75±0.64 to 181.85±2.05 mg/100g and for zinc, 0.60±0.01 to 1.30±0.01 mg/100g . T.C for boiled leaves was between 524.39±9.89 and 1323.5±15.6 µg/g, iron; 95.90±0.01 and 148.75±2.76 mg/100g and zinc 0.60±0.01-1.30±0.01 mg/100g. In vitro bio-accessibility of carotenoids for boiled roots had a highest of 104.42±0.88%, gari had a highest value of 57.22±9.01%, and boiled leaves 0.28±0.01%. Gari recorded the highest frequency of consumption. 36.6% of the respondents had knowledge about yellow flesh cassava. 51.2% were willing to accept yellow flesh cassava. Conclusions: This study has established the T.C, Fe and Zn contents of the new yellow cassava roots and leaves cultivars in Ghana. Fermentation, drying, roasting and boiling retained some carotenoids after processing but solar drying over a long period of time completely degraded carotenoids in yellow cassava roots. Cassava leaves had higher retention of carotenoids but cassava roots had more bio-accessible carotenoids. Carotenoids in yellow flesh cassava leaves had antioxidant properties that have the potential to help combat free radicals in the body. The knowledge and “willingness to accept” yellow cassava was low among Ghanaians. Yellow cassava will serve as a promising source of provitamin A. xii University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 1.0 INTRODUCTION 1.1 Background information Cassava is a relevant root tuber which is a common food for several populations in the tropical and sub-tropical regions of the world (Bradbury and Holloway, 1988). It is the sixth major staple crop after rice, wheat, maize, Irish potato and sweet potato in the world (FAO, 2003). Cassava is a crop which grows on soils with marginal nutrition (Aerni, 2006). Cassava roots contain small amounts of zinc, iron, and β-carotene; however it is the primary basic crop of more than 250 million Africans (Gegios et al., 2010), it was also reported that consumers who often eat cassava are at higher risk for malnutrition especially deficiencies in iron, vitamin-A and zinc than people who consume other diets, particularly those that are predominantly cereal. Micronutrient deficiencies are becoming more endemic specifically in West Africa. The most vulnerable in this population include children, juveniles, and women in their reproductive years, breastfeeding mothers and people who have suffered from war and starvation. Iodine, vitamin A and iron deficiencies are of significant public health importance in several places in Africa and are responsible for the increasing mortality and morbidity in children in Africa (Black et al., 2008). Micronutrient malnutrition, also known as hidden hunger reduces productivity rate and income to individuals and governments in Africa and it also decreases psychological development, decreases working capacity, hinders growth, lowers how immune a person is, causes several pregnancy complications, blindness, goitre and increases the possibility of mortality (Darnton-Hill et al., 2005). 1 University of Ghana http://ugspace.ug.edu.gh The National and Regional estimates of the prevalence of anaemia is 65.7% for children between 6-59 months and 42.4% for women between 15-49 years in Ghana and vitamin A deficiency is also quite high (GHDS,2014) and of public health concern even though there are various intervention strategies being used to combat these micronutrient deficiencies. Cassava is very important to the food security status of most Ghanaian households because it is one of the major staples and it is consumed in various ways such as cooked fresh roots (that include pounded fresh cassava, known as fufu in Ghana), flour (fermented and un-fermented), and granulated roasted cassava (known as gari), fermented pastes known as agbelima (MoFA, 2009). Bio-fortification is the creation of essential nutrient-dense staple crops using the best traditional propagation methods and modern biotechnology (Nestel et al., 2006). Bio- fortification gives a convenient way of getting to people in the rural and remote areas that are malnourished bur do not have access to commercially available fortified (Graham, 1996). A number of staple foods are being fortified. These include rice, sweet potato, maize and cassava. The International Institute of Tropical Agriculture (IITA) with several partners like HarvestPlus over a period of time have developed a number of bio-fortified cassava genotypes popularly known as yellow flesh cassava which contains predominantly β-carotene, a provitamin A. Yellow flesh cassava proposes to be an avenue to solving micronutrient deficiencies in Africa. The International Institute of Tropical Agriculture (IITA) in conjunction with the CSIR-Crop Research Institute of Ghana are currently breeding a number of cassava genotypes in Ghana. 2 University of Ghana http://ugspace.ug.edu.gh 1.2 Problem statement Simler et al., (2005) reports show that vitamin A deficiency causes one in every three deaths of Ghanaian children aged between six and 59 months. Between 2011 and 2020, the number of child deaths attributable to vitamin A deficiency is estimated to be 110,000 (Aguayo and Baker, 2014). To improve vitamin A status among citizens in Ghana, some policies have been employed to reduce vitamin A deficiency prevalence such as food fortification and supplementation with vitamin A tablets, and dietary diversity among the population. Bio fortification is another channel that has been recognised globally as a means to alleviating Vitamin A deficiency in developing countries. As such IITA and its partners Harvest Plus have come out with a bio fortified Cassava yellow flesh cassava. Cassava is a drought resistant crop that can grow on poor soils with little water, can be harvested when needed, providing households with an alternative when the harvest of other crops fails (Aerni P., 2006). Also cassava contains little zinc, iron, and β-carotene, yet it is the primary staple crop of over 250 million Africans. Gegios et al., (2010) reported that frequent consumers of cassava are at greater risk for malnutrition especially deficiencies in vitamin-A, iron, and zinc than consumers of other diets, particularly those that are cereal-based. Clearly then cassava has the potential to pave the way to reducing Vitamin A problems if bio fortified by delivering naturally fortified foods with vitamin A to people with limited access to commercially marketed fortified foods that are more readily available in urban areas (Graham, 1996). This research explored the existing consumption patterns of the white cassava, the knowledge and perception of yellow cassava among cassava consumers in the Greater Accra region of Ghana and also determined the carotenoids, iron and zinc contents of the yellow cassava genotypes in both the roots and the leaves. This will provide information on the 3 University of Ghana http://ugspace.ug.edu.gh contribution of vitamin A from yellow cassava roots and leaves to the daily requirements and inform breeders on the best genotypes. 1.3 Research questions 1. What is the nutritional composition of the newly developed fifteen yellow flesh cassava genotypes (roots and leaves)? 2. Does processing affect the quality of iron, zinc and vitamin A of these yellow flesh cassava genotypes? 3. Do the carotenoids in the yellow flesh cassava genotypes possess antioxidant properties and are they affected by processing? 4. What are the consumption patterns of the white cassava, the knowledge and perception of yellow flesh cassava (processed or unprocessed) among consumers of cassava? 5. What is the contribution of yellow flesh cassava roots and leaves to the RDA of vitamin A? 1.4 Main objective of study The main objective was to determine participants‟ consumption patterns of white cassava, their knowledge and perceptions of the yellow flesh cassava, and the micronutrient composition and antioxidant properties of yellow flesh cassava genotypes. 1.5 Specific objectives The specific objectives of the study were: 1. To determine micronutrient composition (carotenoids, iron and zinc) in the yellow flesh cassava genotypes (fresh roots and leaves, boiled leaves and roots, gari and kokonte). 2. To determine the effect of processing on retention of micronutrient composition (carotenoids, iron and zinc) in the yellow flesh cassava genotypes. 4 University of Ghana http://ugspace.ug.edu.gh 3. To ascertain the in-vitro bio accessibility of the carotenoids in cassava products (boiled leaves and roots, gari and kokonte). 4. To determine antioxidants properties of the carotenoids in yellow flesh cassava genotypes. 5. To assess the consumption patterns of the white cassava and products and as well as perception and knowledge of yellow flesh cassava among consumers of cassava. 6. To determine the contribution of yellow cassava products to the RDA of vitamin A. 5 University of Ghana http://ugspace.ug.edu.gh CHAPTER 2 2.0 LITERATURE REVIEW 2.1 The role of cassava in nutrition Cassava has most of its nutrients in roots and leaves which are the edible parts and is dependent on factors such as terrain, type, age of plants, and climatic conditions (Tewe and Lutaladio, 2004). Cassava roots are predominantly carbohydrate dense (Gil and Buitrago, 2002) and according to Charles et al., (2004), cassava roots are highly concentrated in calories but have little protein, fat, and some minerals and vitamins. Cassava leaves are highly concentrated in vitamins B1, B2, and C, carotenoids, protein and minerals (Adewusi and Bradbury, 1993). Cassava leaves are rich in iron, zinc, manganese, magnesium and calcium (Wobeto et al., 2006). The mineral content of cassava leaves is reported to be two to five times more than that of the cassava roots (Gil and Buitrago, 2002). Cassava leaves have a great amounts of vitamin A in the form of provitamin A carotenoids in comparison to its roots (Gil and Buitrago, 2002). The vitamin A content of cassava leaves is comparable with that of carrots and is higher than those reported for legumes and leafy legumes (Montagnac et al., 2009) 2.1.1 Nutritional problems associated with cassava consumption Cassava consumers have been reported to have high chances of having inadequate vitamin A, zinc and iron intake (Gegios et al., 2010). Consumption of cassava and cassava products containing high quantities of cyanide can lead to acute intoxication, headache, nausea, vomiting, diarrhoea and sometimes death (Akinpelu et al., 2011). 6 University of Ghana http://ugspace.ug.edu.gh 2.1.2 Effects of processing on the nutritional value of cassava Cassava must be processed into different products in order to increase the shelf stability of the products, facilitate conveyance and sales, reduce cyanogenic content and improve palatability and nutrient bio-availability as well (Nyirenda et al., 2011). Cassava processing methods vary, depending on products from simple processing such as peeling, boiling and heating and other complicated procedures for processing (FAO, 2002). For example, gari processing involves peeling, grating, and de-watering, fermenting, sifting, and roasting. Drying is the simplest method of processing cassava (Obilie et al., 2004). Drying causes a reduction in moisture, volume and cyanide concentration of roots, thereby prolonging product shelf life (Westby, 2002). Fermentation is also one of the oldest and most important traditional food processing and preservation techniques (Cardoso et al., 2005). Fermentation is reported by (Achinewhu et al., 1998) to enhance the nutrient content of foods through the biosynthesis of vitamins, fiber digestibility as well as enhancing micronutrient bioavailability. Fermentation also helps in degrading anti-nutritional factors. Thermal processing methods like boiling can also improve the bioavailability of micronutrients such as thiamin, vitamin B6 and carotenoids by discharging them from cells walls in the plant matrix (Erdman & PnerosSchneier, 1994). Processing cassava has been reported to affect the nutritional composition of cassava roots through alteration and losses in essential nutrients (Montagnac et al., 2009). Analysis of nutrient retention shows that raw and boiled cassava root keep the majority of high-value nutrients except riboflavin and iron (Onyenwoke and Simonyan, 2014) 7 University of Ghana http://ugspace.ug.edu.gh 2.1.3 Cassava utilization and consumption in Ghana Cassava is the first crop in terms of energy intake and per capita consumption in Ghana among roots and tubers (Angelucci, 2013), and according to the Food Balance Sheet by (FAOSTAT, 2007), the per capita daily intake is 551 grams which accounts for around 26 % of total per capita daily intake. In terms of calories, cassava consumption per day per person provides 599 kcal (20%) of total daily calorie intake which is 2000 kcal (Angelucci, 2013). Cassava is consumed in various ways across different African countries (Nweke et al., 2002), it also important in the development of the African continent through: being a crop that is draught resistant, rural common food, cash crop for both rural and urban households and, to a minor extent, raw material for feed and chemical industries (Coursey and Haynes, 1970). Cassava is used to prepare products like boiled fresh roots that include pounded cassava, flour and granulated roasted cassava (gari), fermented pastes (agbelima), sedimented starches, and bio-ethanol in Ghana (Ugwu and Ay, 1992). However, Ghana‟s most popular ways of consuming cassava are fufu, gari and flour as well as starch production for the processing industry. Cassava is eaten in all the official regions in Ghana (World Bank, 2010). The study area used in this study was the greater Accra region and (MOFA, 2010) reported a production of 1%. 2.2 The role of vitamin A and carotenoids in human health and nutrition Vitamin A is a fat-soluble vitamin (FAO/WHO, 2002) which exists in three forms namely retinol, retinal and retinoid in animal source foods and provitamin A carotenoids (predominately β-carotene) which are from plant source foods (Wardlaw et al., 2004). The storage form is retinol and is found in the liver until needed by the body (Blomhoff, 1991). Vitamin A is essential for the functioning of the immune system, for good vision 8 University of Ghana http://ugspace.ug.edu.gh (FAO/WHO, 2002). Sommer and West, (1996) reported that improving the vitamin A status of deficient children can improve their disease resistance capacity and thus decrease their mortality and illness from infectious disease significantly. Again, a report by (UNICEF, 1997) stated that enhancing the vitamin A status of children who have a deficiency and are aged 6 months to 6 years expressively increases their chances of staying alive longer. The report also went on to say that the possibility of mortality from measles is decreased by about 50%, from diarrhoea by approximately 40% and overall mortality by 25-35%. Sources of vitamin A include breast milk, other animal milk, liver, eggs, fish, butter, palm oil, mangoes, pawpaw, carrots, orange flesh potatoes, and dark green leafy vegetables (Pan American Health Organization, 2001). Carotenoids are richly coloured molecules and are the sources of the yellow, orange, and red colours of many plants (International Agency for Research on Cancer, 1998). Fruit and vegetables provide most of the carotenoids in the human diet. Carotenoids are also found in some fungi, bacteria and algae (Palace et al, 1999). β-carotene, α-carotene, β-cryptoxanthin, lutein, and lycopene are the most common carotenoids found in human plasma (Rodriguez- Amaya 2004). β -Carotene (Provitamin A carotenoids) is available in green leafy and yellow–orange vegetables and fruits (Veda et al., 2007). The only essential role of carotenoids in humans is that of provitamin A carotenoids which serve as precursors of vitamin A (Chavez et al., 2005), is essential for optimal growth and differentiation of a number of cells and tissues, excellent sight, intellectual development, and general well-being (Njoku et al., 2011). The vitamin A activity of β-carotene in foods is 1/12 that of retinol preformed vitamin A (Institute of Medicine, 2000). 9 University of Ghana http://ugspace.ug.edu.gh Provitamin A also has the advantage of being converted to vitamin A only when needed by the body thus avoiding potential toxicity from an overdose of vitamin A (Delia and Rodriguez-Amaya, 1997). Carotenoids have also been correlated with improvement of the immune system and lowered risk of deteriorating diseases such as cancer, cardiovascular disease, macular degeneration, and cataract (Njoku et al., 2011). 2.2.1 Dietary recommendations for vitamin A and carotenoids Recommended Dietary Allowance (RDA) for vitamin A is dependent on the amount needed to maintain adequate accumulation to support the functions. The recommended dietary allowance for vitamin A for infants and children is 400-600 μg RAE (1,333 -2,000 IU); adolescents (14-18) and adults 19+ is 700 μg RAE (2,333 IU) for females; 900 (3,000 IU) for males, for pregnant women; 750-770 μg RAE (2,333-2,567 IU) with an upper limit (UL) for pregnant women being 3,000 μg RAE or 10,000 IU and for lactating women; 1,200-1,300 μg RAE (4,000-4,333 IU) (IOM, 2000; 2001). The equivalence of vitamin A in foods is measured as retinol activity equivalents. A mixed diet of 12 μg of all-trans-β-carotene or 24 μg of other provitamin A carotenoids (a-carotene, cis β-carotene, β-cryptoxanthin) is equivalent to 1 μg of retinol (Dary and Mora, 2002). 2.3 Role of antioxidants in human nutrition Antioxidants are substances that combat free radicals that cause oxidation of various biomolecules present in an organism (Gan et al., 2010). The human body has an antioxidant protection system, and it has been suggested that a diet highly concentrated in antioxidants strengthens this system (Blomhoff et al., 2006). Free radicals in the body cause cell and tissue damage which is referred to as oxidative damage (Gan et al., 2010). According to Rahmat et al., (2003), the most efficient way to get rid of free radicals is with the support of antioxidant 10 University of Ghana http://ugspace.ug.edu.gh nutrients such as vitamin C (ascorbic acid), vitamin E, and beta carotene (vitamin A) which can be found in large amounts in coloured fruits and vegetables. Carotenoids have also been shown to have some antioxidant properties (Fiedor and Burda, 2014).Carotene reacts with these active free radicals formed during the chain-reaction mechanism of lipid oxidation to form stable inactive products (Burton, 1989). This prevents the oxidative reaction from the production of off-flavours in foods and the potential damage of living cells in biological systems. The antioxidant role played by carotene is independent of its Vitamin A activity (Gomez, 1981). 2.4 Effects of processing on carotenoids Carotene is affected by processing and preservation methods that generally influence the stability of food nutrients (Gomez, 1981). Some traditional food technologies like fermentation and roasting have been reported to have severe effects on carotene (Lyimo et al., 1991). Processing methods such as dehydration, blanching, and canning also have an effect on the antioxidant property of the carotenoids of some dietary plants (Van Het Hof et al., 2000). Vegetables like carrots, lettuce, peppers, potatoes (sweet and Irish) tomatoes and cabbages had increased antioxidant activity upon boiling or steaming (Halvorsen et al., 2006). β-carotene has also been reported to be sensitive to heat and oxidation during blanching and drying (Negi and Roy, 2000). Excessive processing methods such as roasting, prolonged boiling at high temperatures and frying can cause huge losses of provitamin A (Thakkar et al., 2009). However, low heat processing methods such as boiling for a few minutes, soaking, and chopping can improve bioavailability, with little carotenoid loss (La Frano et al., 2013). 11 University of Ghana http://ugspace.ug.edu.gh 2.5 Bio-availability of carotenoids Bio-availability is the percentage of consumed nutrient that is assimilated in the intestine and reaches the point of being absorbed during blood circulation (Maiani et al., 2009). Bio- accessibility refers to the proportion of a carotenoid that is transferred from the food matrix to micelles during digestion and made accessible for intestinal absorption (Stahl, 2000). Bio- conversion is the fraction of bio-available provitamin A that can be changed into retinol (Castenmiller and West 1998). Bio efficacy refers to the efficiency of the process that ingested dietary Provitamin A carotenoids are absorbed and converted to retinol (van Lie shout, 2001).Vitamin A is derived from food as preformed retinol which is readily taken up animal source foods, or as provitamin A from plant source foods which needs to be converted into retinol in the human body (Institute of Medicine, 2001). 2.5.1 Factors that affect bioavailability of carotenoids A number of factors have been reported to influence the bio-availability and bio-conversion of carotenoids. The limiting factors for bioavailability of carotenoids include the quantity of fat available in the food (Thurnham et al., 2003), types of carotene, molecular bonds , amount of carotene in a meal in which the carotenoid is included, nutritional status of the organism, genetic conditions , organism-related conditions ( De Pee et al., 1996). The food matrix also affects the bioavailability of carotenoids as found out by (Parada et al. 2007) who also reported that the structure of fruits and vegetables has a great effect on bio- accessibility and that processing maximizes the release of carotene by disrupting the food matrix. The serum retinol response to ingestion of fruit β-carotene was found to be four times the serum retinol response to ingestion of vegetable β-carotene (De Pee, 1998). Castenmiller and West, (1998) reported that the bioavailability and conversion of carotenoids from foods 12 University of Ghana http://ugspace.ug.edu.gh into retinol is dependent on both food and host-related factors. In green plants, carotenoids are entrapped within chlorophylls (La Frano et al., 2013). Dietary fat has also been shown to raise the bioavailability of carotenoids (Brown et al., 2004). Increasing amounts of dietary fat in the intestine have been suggested to also enhance the formation of micelles by increasing secretion of bile salts, pancreatic lipase and phospholipids, and also enhances chylomicron assemblage by intestinal cells (Borel, 2003). Van Het Hof et al., (2000) reported that the amount of fat required to guarantee carotenoid absorption is three to five grams per meal, even though it is dependent on the physical and chemical traits of the carotenoids consumed. Under limited oil conditions, the micellization of highly lipophilic carotenes (β-carotene and α-carotene) will be inhibited (Liu, 2009). Processing, such as mechanical homogenization or heat treatment has the potential to enhance the bioavailability of carotenoids from vegetables (Van Het Hof et al., 2000; Zhou et al., 2006). Cooking may also enhance the carotenoid release by softening or breaking down the cell walls and by dissociating the protein complex (Rock et al., 1998). Dietary fiber also affects carotenoid bio-availability by interacting with bile acids, reducing the reabsorption of bile acids and fats, and affects the absorption of fat- soluble substances (Liu, 2009). Rock et al., (1998) reported that serum β-carotene content was decreased by 42% when pectin was added to the diet. Unabsorbable fat-soluble compounds reduce carotenoid absorption and interaction among carotenoids may also result in reduced carotenoid bio availability (Van Het Hof et al., 2000). The nutrient status of the host also affects the bio-availability and bio-conversion of carotenoids as reported in a study with vitamin A-replete subjects who after ingestion of a single 40 mg β-carotene dose, had only 22% was absorbed (Novotny et al., 1995). 13 University of Ghana http://ugspace.ug.edu.gh 2.5.2 Estimation of bio-availability of pro-vitamin A. There have been a number of methods in determining the bio-availability of carotenoids. These methods are either in vivo or in vitro. Bio-availability of a food substance is determined in vivo in animals or humans as the plasma concentration of the compound obtained after administration of an acute or chronic dose of an isolated compound or a compound-containing food (Rein et al., 2013). Human studies provide the most applicable results because they are capable of considering host factors, disease states, and physiological changes during digestion (Lee et al., 1999). In vitro models on the other hand, have the potential to provide useful insights about the relative bio-availability of carotenoids from various cultivars of plant foods, and the effects of different styles of processing foods and other components of the meal on the bio-accessibility of ingested carotenoids (Failla et al., 2008). Carbonell-Capella et al.,(2014) reported that the different in-vitro methods used in the assessment of carotenoid bio-accessibility comprised of simulated Gastro intestinal digestion, intestinal segments, brush-border and basolateral membrane vesicles, enterocytes, and transformed intestinal cell lines, mainly Caco-2 human cells. Carotenoids are susceptible to oxidation and thus Garret et al., (2000) added α-tocopherol to his experiment in order to ensure protection against oxidation and improve carotenoids stability. Carotenoids release from the food matrix, the solubility, the measurement and interpretation of plasma response, and the differences in inter-individual response have been reported as some of the challenges to measuring carotenoid bio-availability. (Failla and Chitchumroonchokchai, 2005). 2.6 Methods for determination of total carotenoids There are a number of methods used in the determination of carotenoids in food. These include the spectrophotometric methods, and high performance liquid chromatography 14 University of Ghana http://ugspace.ug.edu.gh (HPLC) methods. Both methods are time consuming, need a laboratory environment and well trained technical operators (Islam and Schwigert, 2014). ICheck™ Carotene on the other hand is a portable photometer and it determines total carotenoid concentration in food and biological fluids. ICheck™ Carotene measures the colour reaction in the test vial and calculates the carotene content in mg/L (Bioanalyt, 2014). 2.7 Methods of beta carotene analysis In recent years, there has been particular emphasis on understanding the types and concentrations of various carotenoids in foods thus the need to quantify beta carotene, the most potent precursor of vitamin A. Methods that are used to determine beta carotene include Gas chromatography (GC), high performance liquid chromatography (HPLC), capillary electro chromatography (CEC), Spectrophotometry and Fourier transform infrared spectroscopy (FTIR) analysis. High performance liquid chromatography is an efficient technique for the detection of β-carotene in various samples and has the ability to discriminate between the similar geometrical forms of carotenoids, a rapid technique and the use of very minute quantity of sample makes it an acceptable method for analysis of different samples (Feltl et al., 2005). Spectrophotometry is an accurate and precise detection method for the quantitative analysis of samples having presence of β-carotene (Zahra et al., 2016). Gas chromatography is a fast and preferred method for the analysis of β-carotene and its cleavage products, focuses on the separation of many volatile mixtures and used to structural and vibration properties of β-carotene (Schlucker et al., 2003). 2.8 Iron and zinc in nutrition and health Iron plays a vital function in the transport of oxygen throughout the body and in cellular processes of growth and division (Beard, 2001). It serves as a coordinated part of necessary 15 University of Ghana http://ugspace.ug.edu.gh enzyme systems in different tissues (Kühn, 1996). Factors that affect iron absorption include the iron status of the person, quantity of dietary haem iron, especially as meat, content of calcium in meal, food preparation (Hallberg et al., 1997). In the human diet, the primary sources of haem iron are the haemoglobin and myoglobin from intake of meat, poultry, and fish whereas non-haem iron is derived from cereals and grains, pulses, legumes, fruits and vegetables (National Health and Medical Research Council, 2006). Iron deficiency is a well- recognized cause of childhood anaemia and has been linked to impaired performance in mental and motor developmental testing, weariness, dizziness, headaches, shortness of breath, ringing in ears, taste imbalances, “restless leg” syndrome, paleness, flattened and inflexible nails, angular stomatitis, glossitis, blue sclera, pica have been reported as some consequences of iron deficiency ( Provan,1999). Zinc is a trace mineral essential to all forms of life as it plays a primary function in gene expression, cell development and replication (Hambridge, 2000). Zinc also helps normal growth and development during pregnancy, childhood, and puberty and is necessary for proper sense of taste and smell (Simmer and Thompson, 1985). Lack of zinc in children can result in stunting and impaired immune response against common infections (Kodkany et al., 2013). In more serious cases, zinc deficiency leads to hair loss, diarrhoea, delay in sexual maturation, infertility, hypogonadism in males, and eye and skin irritations (Maret and Sandstead, 2006). Sea foods, beef, and other meat types which are red are rich sources of zinc; nuts and oil seeds are averagely good plant sources of zinc (King and Cousins, 2006). Recommendations for iron and zinc are shown in the Appendix. 16 University of Ghana http://ugspace.ug.edu.gh 2.8.1 The interrelation of vitamin A, iron and zinc in human metabolism Vitamin A is important in determining the levels of haemoglobin by helping in the release of iron stored in the liver influencing red blood cell production as well as help increase absorption of iron from the intestine (Layrisse et al., 2000; Roodenburg et al., 2000). Iron deficiency can also impair vitamin A metabolism by decreasing vitamin A mobilization from liver stores and perhaps also by decreasing retinol absorption (Jang et al., 2000). Zinc is needed for the synthesis of retinol-binding protein (Ahn & Koo, 1995) thus a deficiency in zinc can then decrease the amount of retinol that can circulate, causing a functional vitamin A deficiency even when the amounts in the liver are sufficient. Zinc is needed by the retinol- binding protein (RBP), which releases retinol, from storage in the liver, one which is similar to the role of iron (Rahman et al., 2002). Conversely, vitamin A is also involved in zinc (Zn) absorption (Wolf et al., 1979). 2.9 Micronutrient deficiencies in West Africa Micronutrient deficiencies have become more widespread specifically in West Africa (Black et al., 2013). The incidence of undernutrition has increased in countries that have experienced economic stress and food insecurity (Dairo and Ige, 2009). The most vulnerable people to undernutrition are children under 5 years of age, adolescents, women of childbearing age, particularly the pregnant and lactating, refugees and victims of famine (Dairo and Ige, 2009). The deficiencies of Vitamin A, iodine and iron are a main public health concern in most parts of Africa and have been the cause of widespread death and sickness in children on the continent of Africa (Black et al., 2008). These deficiencies have caused decreased productivity and loss of revenue to individuals and governments in Africa as well as reduce learning and cognitive ability; impairs growth; reduces immunity; decreases working 17 University of Ghana http://ugspace.ug.edu.gh capacity; causes several pregnancy complications, blindness, and goitre; and raises the risk of mortality (Darnton-Hill et al., 2005). About 75% of children who are from 6 month old to 59 months old are said to be affected with some type of anaemia (GSS/NMIMR/ORC Macro, 2004).More than half of this population are also reported to be affected by the sub-clinical vitamin A deficiency (Micronutrient Initiative, 2004). Reports also show that a deficiency from vitamin A causes 1 out of 3 deaths of Ghanaian minors less than 6 years between 2001 and 2005 (Ghana PROFILES, 2000). 2.9.1 Magnitude and consequences of vitamin A deficiency in Ghana Vitamin A deficiency (VAD) is a major contributor to child mortality and a significant problem in Ghana which affects 26% of the country's under-five population with the middle belt of Ghana also known as the transitional zone reporting 51% of children as having severe and moderate vitamin A deficiency (Agyepong and Amoaful, 1999). Vitamin A deficiency has been defined as a public health problem when more than 15% of the population has serum retinol levels below 0.7 mmol/l (Interdepartmental Committee on Nutrition for National Defence, 1963). Aguayo and Baker (2014) reported prevalence of 15.2 % in 1995 in Ghana. Reports show that vitamin A deficiency causes to one in three deaths of Ghanaian children aged between six and 59 months (Simler et al., 2005). Between 2011 and 2020, the number of child deaths attributable to vitamin A deficiency is estimated to be 110,000 as reported by a survey to estimate Vitamin A deficiency carried out by the Ministry of Health in 1997 (Aguayo and Baker, 2014). The survey found that the prevalence of VAD among children 6- 59 months was highest in the Northern sector of the country (81.2%). The coastal areas had a prevalence of 79.3% and the forest areas had the least prevalence of 73% (Rahman and Agble, 18 University of Ghana http://ugspace.ug.edu.gh 2012). In Ghana, (Lartey, 2009) reported that the most vulnerable are preschool children living in the northern region of the country and women in their reproductive years. (Lartey, 2009) again reported that in the Northern region, the deficiency is a native challenge because there are higher numbers of poor individuals, the climate is mostly rain restricted and the vegetation is savannah. This vegetation comprises of more cereals, grains, nuts and seeds which barely contain vitamin A which makes sources of vitamin A are scarce; hence their use in local food preparation is low. Other parts of the country have plenty sources of vitamin A in the food sources like cocoyam leaves palm nut, mangoes and oranges which are included in the staple food items in that population. Vitamin A deficiency impairs immune function and increases the risk of mortality from diarrhoea, malaria, and measles (West and Darnton-Hill, 2009). It is also the leading cause of child blindness in developing countries. In advanced deficiency, the cornea becomes hazy and can develop erosions, which can lead to its destruction (Sommer and West, 1996). Keratinization of the skin and of the mucous membranes in the respiratory, gastrointestinal and urinary tracts can occur as well as drying, scaling, and follicular thickening of the skin and an increase in susceptibility to respiratory infections (Sommer and West, 1996). 2.9.2 Intervention strategies to combat vitamin A deficiency To improve vitamin A status among citizens in Ghana, some policies have been employed to intervene in vitamin A deficiency prevalence such as food fortification programs that involve fortifying vegetable oils and wheat flour, supplementation with vitamin A tablets, promotion of breastfeeding, yellow flesh sweet potatoes promotion as well as the school feeding programs. 19 University of Ghana http://ugspace.ug.edu.gh 2.9.3 Food fortification programmes in Ghana The objectives of the food fortification programme were to increase vitamin A intake and status among population groups whose daily dietary needs for vitamin A were constituently inadequate , while reducing the risk of excess consumption among groups whose vitamin A status is normal (Dary and Mora, 2002). The minimum level of fortification was determined by setting a target of delivering at least 20% of the Estimated Average Requirement (EAR) for Vitamin A based on the average consumption of the vitamin A foods and these amounts were expected to not have an effect on the sensory properties of the products (Quarshie and Amoaful, 1998). In Ghana, the most common form of vitamin A used to fortify cereal flours is the dry stabilized powder-form vitamin A palmitate, generically referred to as Type 250-SD (75,000 μg RAE/g) (Johnson et al., 2004). This form of vitamin A added to wheat flour to form a premix can remain stable for approximately 15 days, even under hot, humid conditions (Randall and Cubed, 2008). Some challenges with food fortification programs have been that there were technical constraints with installation and maintenance of fortification machinery, as well as the stability of fortificants under the suboptimal distribution and storage conditions traditionally found in developing countries. There are poor distribution systems due to poor infrastructure, lack of access to commercially processed food limited by geography, poverty or cultural preferences, frequent changes of government and a lack of a clearly appropriate food vehicle that addresses all the desirable qualities (Micronutrient Initiative, 1997). 2.9.4 Vitamin A supplementation in Ghana. The induction of higher vitamin A dosage for those who are under five at every six month was on the basis that a single, high dose of vitamin A is well assimilated and stored in the 20 University of Ghana http://ugspace.ug.edu.gh liver, and then reconstituted, when required over a longer period (Ureta et al., 1998). A dose of 100 000 International Units in children from six to eleven months of age and 200 000 IU in children 12–59 months of age was considered to provide adequate protection for 4–6 months, with the exact interval depending on the vitamin A content of the diet and the rate of utilization by the body (Hendricks et al., 1998; USAID/OMNI, 1998).For children aged 6–59 months , a dose of 100 000–200 000 IU of vitamin A is well tolerated, even though side- effects such as headache, nausea or vomiting, and diarrhoea have been reported in 3–7% of these children (Parfitt, 2009). The long-term goal of the vitamin A program in Ghana was to decrease vitamin A deficiency by 80% among children from six months to five years old (Rassas et al., 2004). In Ghana, a national coverage of nearly 90% was obtained during the first functioning vitamin A distribution (MOST, USAID Micronutrient Program, 2004). According to the official data of the Ministry of Health of Ghana, the percentage of children aged 6- 59 months who received vitamin A supplement at least once in Ghana in 2006 was 81.6%. In 2007, the reported coverage was 80% of all children. The vitamin A supplementation in 2011 showed a general decrease with age: about 78% among children aged 6-11 months, and aged 12-23 months, 72% among children aged 36-47 months, and 68% among children aged 48-59 months (MICS, 2011). 2.9.5 Bio-fortification Bio-fortification is the process of breeding nutrients into food crops and it provides a comparatively cheaper, sustainable, and long-term means of delivering more micronutrients (Bouis et al., 2011). Bio-fortification will not only cause a reduction of malnourished people who need treatment by complementary interventions, but also will help them sustain improved nutritional status (Graham et al., 2001). Bio-fortification will also provide a feasible 21 University of Ghana http://ugspace.ug.edu.gh means of reaching malnourished rural populations who may have limited access to commercially marketed fortified foods and supplements (La Frano et al., 2014). Bio-fortified staple foods cannot release as much micronutrients daily as fortified foods or supplements, but they can affect it by raising the daily requirements throughout the life cycle (Bouis et al., 2011). Bio-fortified genotypes are now being acquainted in many countries of Africa. 2.9.5 Bio-fortification of crops in Africa. Bio-fortification can be achieved either by genetic modification or by selective breeding (Tiwari et al., 2010). Bio-fortification by genetic modification is done by moving useful genes from one organism‟s chromosomes into the cells of another and bio-fortification by selective breeding is when the best plants or animals are bred together to get the best possible offspring. Bio- fortification of crops in Africa is done mostly by selective breeding (Manshardt, 2004). Some crops that have been bio-fortified in Africa include millet, maize, sweet potato, rice, sorghum and cassava (Bouis et al., 2009) and the nutrients that are targeted during bio- fortification include vitamin A, lysine, iron and zinc (Saltzman et al., 2013). The current research studies on using plant Provitamin A carotenoids in humans have shown promising results. One example of this is the use of orange-fleshed sweet potato (OFSP) to prevent vitamin A malnutrition and this has been used in a few feeding trials in some countries in Africa and Asia. Through conventional breeding, β-carotene contents in the orange-fleshed sweet potato could reach as high as 194 μg/g (Low et al., 2007). Bio-fortified maize adequately maintained vitamin A status in Mongolian gerbils and was as efficacious as β- carotene supplementation (Howe and Tanumihardjo, 2006). Each serving of 125g per day for 53 school days to 5–10 year old children in Durban, South Africa showed significant improvement of vitamin A status (Van Jaarsveld et al., 2005). Even though strategies have 22 University of Ghana http://ugspace.ug.edu.gh been put in place to combat Vitamin A deficiency, there is more room for improvement thus the introduction of bio fortified cassava. 2.9.6 Bio-fortification of cassava Yellow flesh cassava is reported to have originated from South America (Iglesias et al., 1997). Yellow pigmented cassava is reported to be cultivated in a limited way in Colombia, Philippines, Jamaica and some African countries (Vimala et al., 2009). The International Institute of Agriculture (IITA) created cultivars with increased β-carotene concentrations through natural plant-breeding methods and introduced them in Nigeria (Chavez et al., 2005). Yellow cassava genotypes that have been cultivated in Nigeria in 2011 were reported to include 6 μg/g (fresh weight) total β-carotene, however breeding is currently underway to deliver cultivars up to 15 μg/g of β-carotene (fresh weight). Yellow flesh cassava has colours ranging from a deeper shade of yellow to lighter shade of orange (Saltzman et al., 2013). Bio- fortification alters the colour of cassava roots to deep yellow due to the increase in provitamin A content as well as the taste which can be influenced due to lower dry matter concentration associated with higher provitamin A concentration (Chavez et al., 2005). 2.9.7 The nutritional value of yellow flesh cassava Beta carotene is the significant carotenoid in cassava, but is found as a combination of the Trans and cis-forms (Rodriguez-Amaya and Kimura, 2004). Esuma et al., (2012) found that the total carotenoid content in cassava roots ranged from 1.2 to 14.2μg/100 g fresh tissue, implying that they can be a significant source of provitamin A pigments for human populations deprived of them. Even though yellow cassava is a splendid source of β-carotene and energy, it is reported to be generally poor in other micronutrients such as zinc and iron (Maziya-Dixon et al., 2000). The provitamin A carotenoid in bio-fortified cassava is primarily 23 University of Ghana http://ugspace.ug.edu.gh β-carotene. Total carotene levels for fresh yellow cassava roots were reported to be between 6.2–7.8 µg/100g (fresh weight) (Ceballos et al., 2012). Ukenye et al., (2013) also reported proteins, fat as well as fibre for the yellow cassava roots genotypes . The leaves of yellow cassava were also reported to be rich in carotene reaching the levels of 13,000 to 16,000 I.U (Akinwale et al., 2010). However, high moisture contents between 60-80% have been reported for yellow cassava roots. 2.9.8 Effects of processing on the carotenoid retention of yellow flesh cassava. The effect of storage on carotenoid in products made from bio-fortified crops has also been reported by a number of studies. Mugode et al., (2014) reported on the sustenance of carotenoids during storage of bio-fortified maize and it was shown that majority of the carotenoid breakdown occurred in the first few weeks of storage and the degradation rate then slowed down. Bechoff et al., (2011) who worked on bio-fortified sweet potato similarly reported that storage of dried potato chips had a dramatic effect on carotenoid sustenance which was about 80% loss in about 4 months. Processing technologies such as steaming or boiling before dehydration has also been established to enhance the carotenoids that are retained in dehydrated foods by preventing enzymes that can cause carotenoids degradation from functioning (Koca et al. 2005). Gari, a product of cassava processed through fermentation and gelatinization, has been reported to have β-carotene retention of about 32%. A 90% decrease of β-carotene content was however reported after 20 minutes fermentation and roasting at 195°C (Thakkar et al., 2009). Chavez et al., (2007) measured the carotenoid retention during a four-week of storage for yellow flesh cassava flour and chips that had been dried (oven or sun). The β-carotene retention of yellow flesh cassava dropped from 72% upon oven-drying to 40% and 32% after two and four weeks of storage at room temperature, 24 University of Ghana http://ugspace.ug.edu.gh respectively. The reduction rate for the sun- dried products was more severe. In that process, the β-carotene retention which was 38% further reduced to 24% and 18% after two and four weeks of storage, respectively. Greater losses in total carotenoid content have been reported for cassava flour stored at the same temperature in a dark place (Oliveira et al., 2010). Other methods, including boiling, oven-drying and sun-drying all reduced β-carotene retention at different levels, ranging from 20 to 90% (La Frano, 2013). Leaching in water and heat did not adversely affect the carotene content during processing, but drying in the sun (ultraviolet light) had detrimental effect on the production of provitamin A fufu (Omodamiro et al., 2012). Retention varies between 10% for cassava products that have been processed to a greater extent and for products like gari and 87% for boiling (Maziya-Dixon et al., 2000; Thakkar et al., 2009). 2.9.9 Bio-availability of carotenoids in yellow flesh cassava Bio-availability of carotenoids from plant based foods have been reported from a number of studies as being limited as a result of phytochemicals and other anti-nutrient factors (Gibson, 1994; West et al.,2002). Bouis et al., (2003) reported that cassava carotenoids can provide up to 25% of the estimated average requirements (EAR) for women and preschool children. Consuming yellow cassava has been shown in a study to have small but significant improvements in vitamin A status of children (Talsma, 2014). Phorbee et al., (2013) reported 11 to 18% bioavailability of carotenoids in gari made from yellow cassava when fed to rats. The study concluded that cassava β-carotene could maintain rat growth and avoid vitamin A deficient symptoms. Another study by La Frano et al., (2013) showed that bio-fortified cassava increases β-carotene and retinyl palmitate TAG-rich lipoprotein plasma 25 University of Ghana http://ugspace.ug.edu.gh concentrations in healthy well-nourished adult women, suggesting that it is a potential intervention food for preventive measures against vitamin A deficiency. 2.9.10 Consumer acceptability, perceptions and knowledge on bio-fortified crops During bio-fortification, specific fortificants are introduced into essential crops through breeding. There are usually changes in the colour and sensory properties of this food crop aside its increased nutritive value. According to (Tumuhimbise et al.,2013), these replacements play a part in whether the bio-fortified crops are accepted by the intended consumers or not. The acceptability of bio-fortified crops is affected by several factors. The method of breeding has been one of the issues for communities to reject certain provitamin A crops (Tumuhimbise et al., 2013). Bio-fortification as stated earlier can be achieved through conventional selective breeding or through genetic engineering (Tiwari et al., 2010). However, genetic engineering as a method for bio-fortification has been damaging. In Europe for example, genetically modified crops have been accepted with uncertainty because of concerns raised by consumers about the risk of getting harmed in relation to human health, to the environment, and the concern about the „unnatural‟ status of the process of bio- fortification (Nuffield Council on Bioethics, 1999). Again, in the case of bio-fortified crops where nutrients are visible such as it is in provitamin A crops, the colour of the crops becomes a vital issue in its acceptance and consumption (Van Jaarsveld et al., 2005). Nutrition education is essential in communicating the nutritional and health benefits of bio fortified crops. Nutrition education has also been cited as an important factor that affects acceptability of bio-fortified crops (Tanumihardjo, 2008). Chowdhury et al., (2011) reported that mothers in Uganda easily adopted bio-fortified foods after receiving nutrition education. Consumer preferences for bio-fortified foods have also been influenced by providing 26 University of Ghana http://ugspace.ug.edu.gh nutritional information in Ghana (Groote et al., 2010). When given the same information, “willingness-to-pay” research for orange flesh potato, maize, and yellow flesh cassava showed that consumers liked the organoleptic traits of the bio-fortified crops and will give out money for a higher payment for high provitamin A cultivars than for white genotypes (Chowdhury et al., 2011; Meenakshi et al., 2012; Oparinde et al., 2012). 27 University of Ghana http://ugspace.ug.edu.gh CHAPTER 3 3.0 METHODOLOGY This study was in two parts; the first section was laboratory analysis to quantify the total carotenoids, iron and zinc contents in fresh cassava leaves, roots and cassava products (gari, konkonte, boiled cassava roots and leaves) as well as the antioxidant activity and the in vitro bio accessibility of the carotenoids. The second section was a cross sectional study survey to identify the consumption patterns of white cassava, the knowledge and perception of yellow flesh cassava among Ghanaians. 3.1 Laboratory study The laboratory study comprised moisture analysis, total carotenoids, iron and zinc determination, antioxidant activity and in vitro bio-accessibility of carotenoids. 3.1.1 Sample collection and preparation Fourteen yellow flesh cassava genotypes were planted by IITA in Crops Research Institute (CRI) at Pokuase in the Greater Accra Region of Ghana. Moisture contents, total carotenoids, iron and zinc determination was done for all fourteen genotypes before processing. Out of these fourteen genotypes, five were selected by IITA to study the effects of processing on the total carotenoid retention, beta carotene concentration, in-vitro bio accessibility, and iron and zinc concentration of the cassava genotypes. Reasons for the selected five were because previous trials concluded that these genotypes (01, 03, 05, 07, and 15) had sensory and textural characteristics preferred by farmers as well as high contents of total carotenoids. Yellow flesh cassava samples were collected at the Crops Research Institute (CRI) at Pokuase. Samples were processed and bagged in the Nutrition and Food Science Department, University of Ghana, Legon. 28 University of Ghana http://ugspace.ug.edu.gh 3.1.2 Preparation of gari Cassava roots were peeled, washed and grated. The grated cassava was put into perforated jute bags, fermented for 24 hours and dewatered. The grated cassava was then sifted over a raft and roasted over minimal heat till crispiness was achieved to avoid loss and degradation of carotenoids in the cassava. Temperature and lighting were not strictly controlled to mimic the garification methods used among gari processors in Accra and also to quantify carotenoids are lost during the process. The resulting product, gari, was allowed to cool and packaged in an air tight bag and stored in cool dark place to help prevent carotenoid loss and degradation. 3.1.3 Preparation of kokonte The cassava roots were harvested from the farm and peeled. The peeled roots were washed and chopped into bigger chunks. The chopped roots were dried evenly in a solar dryer for 5 days after which they were milled and packaged in black polythene bags and stored in a cool dry place for further analysis. 3.1.4 Preparation of boiled roots and leaves Fresh roots and leaves samples were chopped separately into small pieces and 5 grams weighed into a plastic bag and boiled in a stainless steel pan on an electric cooker for fifteen minutes and cooled for further analysis. 3.1.5 Determination of moisture content Moisture content was determined by weighing 2g of sample (fresh leaves and roots, gari, kokonte, boiled leaves and roots separately) and dried in an air oven at a temperature of 105°C for 24 hours. The samples were removed, cooled over a desiccant for 15 minutes, weighed 29 University of Ghana http://ugspace.ug.edu.gh and the loss in weight of sample after drying was calculated as percent moisture (AOAC, 2005). 3.1.6 Quantification of Beta carotene in yellow cassava genotypes Beta carotene in yellow cassava genotypes and their products (fresh roots and leaves, gari, kokonte, boiled leaves and roots) were assessed using high performance liquid chromatography (HPLC). Five grams (5g) of each sample was weighed on a digital balance (Professional electronic balance) into a mortar and ground with pyrogallol to prevent oxidation of the carotenoids. The carotenoids were extracted by successive additions of 25 mL of acetone to the weighed sample and the final solution transferred into a sintered funnel (5μm) coupled to a 250 mL Buchner flask and filtered under vacuum. This procedure was repeated three times or until the sample became colourless. The extract obtained was transferred to a 500 mL seperatory funnel containing 20ml of petroleum ether. The acetone was removed through the slow addition of ultrapure water to prevent emulsion formation and the aqueous phase was discarded. This procedure was repeated four times until no residual solvent remained. The extract was then transferred through a funnel to a 50 mL volumetric flask. The sample was evaporated with a rotary vacuum evaporator to dryness. The concentrate was reconstituted with 2ml of acetone and put into vials for HPLC analysis (Rodriguez-Amaya and Kimura, 2004). 3.1.7 Determination of total carotenoids in fresh yellow cassava roots The iCheck ™ carotene measures total carotenoids in vitamin premix, food and biological fluids. iCheck Carotene measures the colour in the test vial and calculates the total carotenoid content in mg/L. The principle of the method is photometric and its sample volume per analysis is 400 µL (0.4 mL). It works in a linear range of 0.15–25.0 mg/L. Time per 30 University of Ghana http://ugspace.ug.edu.gh TM analysis for the device is < 2 min and its variation is < 10%. ICheck carotene has been validated against HPLC (published in Vet. Clin. Pathol., 2011 (Bioanalyt, 2014). 3.1.7.1 Fresh yellow cassava roots About 5g of the fresh yellow cassava roots was weighed and ground to a smooth paste to help solubilize all the carotenoids present in the sample and transferred into a falcon tube and made to 25ml mark with distilled water. 0.4ml of the diluted sample was injected into the reagent vial. The vial was vigorously shaken for 10 seconds till the content of the vial appeared as one uniform solution. The vial was made to stand still for 5 minutes until the solution in the vial appeared as two distinct phases. The vial with the sample was measured with iCheck™ Carotene earliest 5 minutes and latest 1 hour after sample injection. The total carotenoid was calculated by multiplying the iCheck™ reading with the dilution factor (Bioanalyt, 2014). 3.1.7.2 Fresh yellow cassava leaves The procedure was modified for the leaves because it contained excess amount of carotenoids which was above the working range of the icheck device thus 0.5g of the fresh yellow cassava leaves was weighed and ground to a smooth paste and transferred into a falcon tube and made to 50ml mark with distilled water. 0.4ml of the diluted sample was injected into the reagent vial. The vial was vigorously shaken for 10 seconds till the content of the vial appeared as one uniform solution. The vial was made to stand still for 5 minutes until the solution in the vial appeared as two distinct phases. The vial with the sample was measured with iCheck™ Carotene earliest 5 minutes and latest 1 hour after sample injection. The total carotenoid was calculated by multiplying the iCheck™ reading with the dilution factor (Bioanalyt, 2014). 31 University of Ghana http://ugspace.ug.edu.gh 3.1.7.3 Kokonte made from yellow flesh cassava Two grams (2g) of the sample was weighed and ground to a smooth paste and transferred into a falcon tube and made to 30ml mark with distilled water. 0.4ml of the diluted sample was injected into the reagent vial. The vial was vigorously shaken for 10 seconds till the content of the vial appeared as one uniform solution and the vial made to stand still for 5 minutes until the solution in the vial appeared as two distinct phases. The vial with the sample was measured with iCheck™ Carotene earliest 5 minutes and latest 1 hour after sample injection. The total carotenoid was calculated by multiplying the iCheck™ reading with the dilution factor (Bioanalyt, 2014). The procedure was modified for kokonte because it had a high swelling capacity which blocked the syringe during the injection into the vials of the icheck device. 3.1.7.4 Yellow cassava gari The procedure was modified for gari because it had a high swelling capacity which blocked the syringe during the injection into the vials of the iCheck device thus two grams (2g) of the sample was weighed and ground to a smooth paste and transferred into a falcon tube and made to 30ml mark with distilled water. 0.4ml of the diluted sample was injected into the reagent vial. The vial was vigorously shaken for 10 seconds till the content of the vial appeared as one uniform solution and the vial made to stand still for 5 minutes until the solution in the vial appeared as two distinct phases. The vial with the sample was measured with iCheck™ carotene earliest 5 minutes and latest 1 hour after sample injection. The total carotenoid was calculated by multiplying the iCheck™ reading with the dilution factor (Bioanalyt, 2014). 32 University of Ghana http://ugspace.ug.edu.gh 3.1.8 Determination of iron and zinc contents Iron and zinc contents in yellow cassava products (fresh and boiled roots and leaves, gari and kokonte) were assessed using a method by AOAC (2005). Wet digestion was used to digest samples. A weight of 0.1g of each sample (fresh and boiled roots and leaves, gari and kokonte) was digested in 4ml of concentrated sulphuric acid and heated in a fume hood. 10 drops of H2O2 was added to the digest and heated from black to dark brown. Six drops of hydrogen peroxide (H2O2) were added and heated till the digest was colourless. It was then transferred to a 100ml volumetric flask and made up to the 100ml mark with deionized water. The wet digest solutions were prepared on the same day for each sample as the check sample and duplicate blanks. Iron and zinc contents were determined by the Atomic Absorption Spectrophotometer (Analyst 400 Perkin Elmer). 3.1.9 Determination of antioxidants properties of carotenoids in yellow flesh cassava Antioxidant assays were based on the measurement of the loss of 2, 2-Diphenyl 1, 1- picrylhydrazyl (DPPH) colour at 515 nm after reaction with test samples and the reaction is monitored by a spectrophotometer. The percentage of the DPPH remaining was proportional to the antioxidant concentration of the test samples .One gram (1g) of each sample (fresh and boiled roots and leaves, gari and kokonte) was ground and carotenoids were extracted using 50ml of 95% methanol as solvent. The solution was filtered using a Whatmann filter paper (grade F101 with pore size of 125mm) till the residue was white. 3ml of the filtrate was added to 3ml of DPPH, vortexed and allowed to stay in the dark for 30 minutes. The absorbance was read using the Atomic absorption spectrophotometer (T80 UV/VIS spectrophotometer-PG instruments Ltd) using a wavelength of 450nm (Bondet et al., 1997). 33 University of Ghana http://ugspace.ug.edu.gh 3.1.10 Determination of carotenoid retention after processing For the estimation of true carotenoid retention, the following formula was used: % True Retention = c1 × wb × 100 c2 × wub Where c1 is the carotenoid content in processed sample, c2 is the carotenoid content in fresh (unprocessed) sample, wb is the weight of processed sample, and wub is the weight of fresh (unprocessed) sample. Carotenoid content (µg/g) was calculated on dry matter basis for both the processed and unprocessed root samples (Murphy et al., 1975). 3.1.11 Determination of in vitro bio-accessibility of carotenoids in yellow flesh cassava The method was adapted from Hedre´n, et al., (2002) with some modifications. Three grams (3g) of the cassava sample was weighed and ground in a mortar. For the oral phase of the digestion, 0.003grams alpha amylase was added to 0.005M saline solution (NaCl). About 7.5 ml of the saline solution was added to sample and sonicated in a water bath at a temperature of 37ºC and pH of 6.8. After 10 minutes, the sample solution was acidified to pH of 3 using 0.01M HCl to stimulate the gastric phase. 1ml of porcine pepsin (40mg/ml) was added to the sample solution at pH 3 and incubated for an hour at 37ºC after which pH was brought up to 7 to stimulate the intestinal phase of the digestion. About 4.5 ml of a mixture of porcine pancreatin (4mg/ml) and porcine bile extract (24mg/ml) was added to the stimulated gastric fluid and 1% (w/v) alpha tocopherol. The final volume was adjusted to 25ml with physiological saline solution and incubated for two hours in a shaking water bath (100rpm at 37ºC). It was then centrifuged at 5000g for 45minutes at 4ºC. The supernatant was collected with a plastic pipette into a seperatory funnel containing 20ml of 25% (w/v) NaCl. 20ml of 95% ethanol and 15ml of petroleum ether were added and allowed to separate. The solution 34 University of Ghana http://ugspace.ug.edu.gh was washed with water and the organic phase collected and the absorbance read with an atomic Absorption spectrophotometer at 450nm. 3.1.12 Contribution of yellow cassava to recommended daily allowances for under 5 children Contribution of yellow cassava to the recommended daily allowance for vitamin A was calculated first using a conversion factor of 1µg/g of beta carotene =3.3IU of vitamin A(Bioanalyt,2015) to get the amount of vitamin A in the food . After that, An example for one of the sample is shown below: 1µg/g of beta carotene in food sample =3.3IU of vitamin A 3.439ug /g of food sample =3.3IU *3.439 = 11.348 IU Thus if every 1g of food contained 11.34IU of vitamin A, then an allowance of 200IU which is the upper limit will be: =2000/11.35 =176g of food Thus for every 100grams = (2000*100)/176 =1136.36 IU To find the %RDA= (1136.36/2000)*100 This was also done using the lower limit and an average to give the final % RDA 35 University of Ghana http://ugspace.ug.edu.gh 3.2 Cross sectional study A survey was conducted to describe the consumption patterns of white cassava and the perception, knowledge of participants on yellow flesh cassava. A pretested semi-structured questionnaire was used to interview adult Ghanaians. Data on their socio demographic characteristics were also collected. 3.2.1 Study area and population The study area selected in the Greater Accra region and was chosen for convenience and also because the cassava genotypes were planted in Greater Accra as well. The study population comprised individuals who were above 18 years of age and consumed cassava. This population that was chosen was representative of the cassava consumers. 3.2.2 Sample size determination and sampling technique for the survey Simple random sampling was employed in subject selection for the survey and a sample size of 287 was used based on the formulae by 2 n0= Z pq 2 E n0= sample size, Z= confidence interval (1.96), p=α level(0.5), q=1-p, E= margin of error( 0.05) (Cochran, 1963). 3.2.3 Data collection Data were collected on background information, consumption and usage of cassava, knowledge and the perception of yellow flesh cassava using a pretested questionnaire. 36 University of Ghana http://ugspace.ug.edu.gh 3.2.3.1 Socio-demographic characteristics Data were collected on participants‟ age, marital status, educational level, occupation, residential status, income and household size. 3.2.3.2 Consumption and usage of white cassava Data were collected on the frequency of cassava and cassava products consumed, time of consumption, reasons for cassava consumption and knowledge on the nutritive value of white cassava. 3.2.3.3 Knowledge and perception of yellow flesh cassava Participants were interviewed on knowledge about yellow cassava, source of information about yellow flesh cassava, willingness to accept yellow flesh cassava and perceived usage of yellow cassava. 3.3 Data analyses The data analysis tools used were Statistical Package for Social Sciences version 16(SPSS), MINITAB version 17, Statistical Analysis System version 9.01 (SAS) and Microsoft Excel. 3.3.1 Laboratory data Means and standard deviations were computed using Microsoft Excel. Analysis of variance was used to compare means for laboratory analyses. 3.3.2 Survey data Descriptive statistics were computed using SPSS version 17 for demographic data and consumption patterns of knowledge on yellow cassava. Logistic regression was computed using Statistical Analysis System version 9.01. Multinomial regression was used and this provided the opportunity to see the interaction between more than two categories of an outcome (knowledge and „willingness to accept‟ yellow flesh cassava) and predictors (in this 37 University of Ghana http://ugspace.ug.edu.gh case, selected socio-demographic factors). The socio-demographic factors; education status, marital status, age and gender were considered as the independent variables. 3.4 Ethical Consideration Ethical clearance was sought from the College of Basic and Applied Science (CBAS) ethical clearance board. Informed consent was also sought from all participants interviewed. 38 University of Ghana http://ugspace.ug.edu.gh CHAPTER 4 4.0 RESULTS 4.1 Preamble This study was primarily in two sections; laboratory analysis and a survey. The laboratory analysis focused on determining the total carotenoids, iron and zinc of fourteen yellow flesh cassava genotypes. The effect of processing on the nutrients and the antioxidant activity of the carotenoids present in the roots and leaves of the selected yellow cassava genotypes were also determined. The same analysis was carried out on some selected yellow cassava products (gari, boiled cassava roots, leaves and kokonte). The breeder‟s names of the cassava genotypes are in Appendix III. The survey provided information on the consumption patterns of white cassava among Ghanaian consumers and their willingness to accept the yellow flesh cassava. The survey also determined associations between socio-demographic characteristics and consumption patterns. 39 University of Ghana http://ugspace.ug.edu.gh 4.2 Laboratory analysis Fourteen yellow flesh cassava genotypes were planted by IITA in Crops Research Institute (CRI) at Pokuase in the Greater Accra Region of Ghana. Moisture contents, total carotenoids, iron and zinc determination was done for all fourteen genotypes before processing. Out of these fourteen genotypes, five were selected by IITA to study the effects of processing on the total carotenoid retention, beta carotene concentration, in-vitro bio accessibility, iron and zinc concentration of the cassava genotypes. Iron, zinc and carotenoids contents were reported on dry matter basis. 4.2.1 Total carotenoids, iron and zinc contents of fresh yellow cassava roots Fresh yellow cassava roots had moisture content between 67.05±1.09% (sample 08) and 82.123±0.76% (sample 15) (Table 1). Total carotenoids ranged from 4.73±0.11 µg/g (sample 07) to 10.11±0.18µg/g (sample 09). Iron content for the samples ranged from 87.35±3.18 to 146.25±1.20 mg/100g (sample 06 and 03) and for zinc, 0.30±0.01 to 1.55±0.07 mg/100g (sample 06 and 14). There were significant differences among all samples (p<0.05) with respect to dry matter, total carotenoids, iron and zinc. Table 1: Total carotenoids, iron and zinc contents of fresh yellow flesh cassava roots (d.m.b) Sample Moisture content Total Iron Zinc (%) carotenoid (mg/100g) (mg/100g) (µg/g) abc d g def 01 78.30±0.96 7.85±0.07 76.10±1.27 0.45±0.07 bcde g d bcdef 02 76.17±0.25 4.67±0.03 98.70± 0.85 0.70± 0.14 efgh f a bcde 03 71.60±1.15 5.77±0.07 146.25±1.20 0.85±0.070 gh f d bc 05 68.40±1.74 5.69±0.04 112.20±0.14 0.95±0.07 abc g f f 06 79.08±0.32 5.02±0.07 87.35±3.18 0.30± 0.01 bcd g d cdef 07 76.21±1.04 4.73±0.11 111.00±0.42 0.60±0.14 40 University of Ghana http://ugspace.ug.edu.gh h e b cdef 08 67.05±1.09 6.57±0.28 131.05±1.06 0.60±0.14 cdef a f ef 09 75.55±1.99 10.11±0.18 88.65±0.21 0.40 ±0.01 gh b c bc 10 70.22±0.57 9.21±0.14 121.90±0.01 1.05±0.07 fgh e c bcd 11 71.39 ±2.32 6.46±0.04 123.85±2.76 0.90±0.01 defg e a bcdef 12 72.76±0.42 6.43±0.14 143.45±2.33 0.70±0.28 ab c c ab 13 80.33±0.58 8.43±0.07 121.55 ±0.21 1.10±0.14 fgh ef bc a 14 70.98±0.19 6.16±0.04 126.15±0.49 1.55±0.07 a d d bcdef 15 82.12 ±0.76 7.69±0.04 110.25±0.07 0.70±0.01 P-value 0.00 0.00 0.00 0.00 Means with different superscript on the same column are significantly different at P ≤ 0.05. 4.2.2 Total carotenoids, iron and zinc contents of fresh yellow cassava leaves The moisture content was between 56.06±0.33% (sample 09) and 81.62±0.52% (sample 10) (Table 2). T.C ranged from 792.93±0.98 µg/g (sample 08) to 2649.20±29.10 µg/g (sample 07). Iron and zinc values ranged between 118.35±0.07 and 182.05±0.07 mg/100g (sample 08 and sample 01) and 3.75±0.64 and 15.50±0.14 mg/100g (sample 08 and 10). There were significant differences among all samples for each parameter measured (p<0.05). Table 2: Total carotenoids, iron and zinc contents of fresh yellow cassava leaves (d.m.b) Sample Moisture Total Iron Zinc content (%) carotenoids (mg/100g) (mg/100g) (µg/g) cde b a h 01 63.65±0.89 2224.51±2.84 182.05±0.07 6.85±0.07 ef b 02 63.65±0.89 2223.80±28.90 ab def 147.20±0.28 8.95±0.07 f g 03 61.82±0.31 862.20±21.50 a gh 177.3±46.60 7.80±0.70 b c ab bc 05 70.69±0.55 2034.74±8.23 147.6±25.00 13.42±0.16 b d f efg 06 69.70±0.49 1798.76±2.76 163.85±1.34 8.55± 0.07 bcd a 07 67.37±0.58 2649.20±29.10 d i 181.55±0.63 4.25±0.21 41 University of Ghana http://ugspace.ug.edu.gh f g 61.01±1.43 792.93±0.98 b i08 118.35±0.07 3.75±0.64 g a 09 56.05±0.32 2593.90±101.80 a c 181.75±0.64 12.75±0.07 a b 10 81.62±0.52 2312.53±12.56 ab a 143.40 ±0.14 15.50±0.14 f f 11 60.79±1.40 991.85±8.86 ab d 165.25±0.35 9.75± 0.07 bc c 12 68.21±1.40 2042.74±1.86 ab fg 152.90±0.14 8.25±0.07 ef d 13 62.60±0.45 1810.28±4.86 ab gh 174.40±0.42 7.50±0.28 cde e 14 65.92±0.10 1171.15±0.95 ab de 168.10±0.14 9.40±0.01 def e 64.12±1.05 1133.16±1.92 ab b15 129.65±0.21 14.30±0.14 P- 0.00 0.00 0.00 0.00 value Means with different superscript on the same column are significantly different at P ≤ 0.05. 4.2.3 Total antioxidant activity of fresh yellow cassava roots and leaves Total antioxidant activity of fresh roots (Table 3) ranged from non-detectable (ND) to 86.28±0.10% (sample 02). Fresh leaves had total antioxidant activity between 17.39±0.10% for (sample 03) and 70.96±0.10% (sample 12). The antioxidant activity was also expressed in Alpha Tocopherol units. For fresh leaves, antioxidant activity ranged from 13.64±0.05 Alpha Tocopherol units (sample 12) to 38.81±0.05 Alpha Tocopherol units (sample 03). For cassava roots, values ranged between ND to 8.53±0.05 Alpha Tocopherol units (sample 02). There were significant differences among all samples for each parameter measured (p<0.05). 42 University of Ghana http://ugspace.ug.edu.gh Table 3: Total antioxidant activity of fresh yellow cassava roots and leaves (d.m.b) Sample Fresh roots Fresh roots Fresh leaves (%) Fresh leaves (%) (AT units ) (AT units) a a b m 01 81.85±0.10 8.53±0.05 62.45 ±0.10 17.64 ±0.05 b b e j 02 86.28±0.10 6.44±0.05 51.03±0.01 23.00±0.01 c c n a 03 *ND *ND 17.39±0.10 38.81±0.05 c c m b 05 *ND *ND 26.31±0.27 34.61±0.13 c c f i 06 *ND *ND 50.44±0.21 23.28±0.10 c c j e 07 *ND *ND 41.63±0.01 27.42±0.01 c c l c 08 *ND *ND 37.67±0.27 29.28±0.13 c c d k 09 *ND *ND 57.13± 0.10 20.14±0.05 c c g h 10 *ND *ND 48.20±0.01 24.33±0.01 c c h g 11 *ND *ND 44.47±0.01 26.08±0.01 c c a n 12 *ND *ND 70.96±0.10 13.64±0.05 c c k d 13 *ND *ND 38.97±0.18 28.67±0.08 c c i f 14 *ND *ND 43.64±0.10 26.47±0.05 c c i l 15 *ND *ND 59.85±0.20 18.86±0.10 P-value 0.00 0.00 0.00 0.00 *ND means non detectable, Means with different superscript on the same column are significantly different at P ≤ 0.05. AT- alpha tocopherol units 43 University of Ghana http://ugspace.ug.edu.gh 4.2.4 Total carotenoids, iron and zinc contents of yellow flesh cassava products Five out of the fourteen genotypes were selected based on farmer preferences and previous trials by IITA to further study the effect of processing on total carotenoids, iron and zinc content, antioxidant activity, in-vitro bio accessibility and beta carotene concentration. The selected genotypes were 01, 03, 05, 07, 15 and were made into gari, kokonte, boiled and boiled leaves 4.2.4.1 Total carotenoids, iron and zinc contents of yellow flesh gari Dry matter content was between 94.74±0.07% and 97.12±0.09% for samples 05 and 07 respectively. Total carotenoids were between 7.39±1.06 µg/g (sample 07) and 3.21±2.79 µg/g (sample 05). Iron content for the samples ranged from 118.75±0.64 to 181.85±2.05 mg/100g (sample 07 and 15) and for zinc, 0.25±0.07 to 0.80±0.14 (sample 01 and 15). There were significant differences among all samples (p<0.05) with respect to dry matter, total carotenoids, iron and zinc (Table 4). Table 4: Total carotenoids, iron and zinc contents of yellow flesh gari (d.m.b) Sample Moisture Dry matter Total Iron Zinc content (%) (%) carotenoids (mg/100g) (mg/100g) (g/100g) c b a 01 3.36±0.11 96.64±0.12 7.37±0.51 c b 143.40±0.57 0.25±0.07 c b ab 03 3.19±0.02 96.81±0.02 6.00±0.45 b a 156.10±1.13 1.10±0.14 a d b 05 5.25±0.07 94.75±0.07 3.21±2.79 d a 125.50±0.28 0.80±0.01 d a a 07 2.88±0.09 97.12±0.09 7.39±1.06 e b 118.75±0.64 0.35±0.07 b c a 4.01±0.08 95.99±0.08 6.96±0.33 a15 181.85±2.05 0.80±0.14a P- 0.00 0.00 0.00 0.00 0.00 value P-value <0.05; Means that do not share a letter are significantly different. 44 University of Ghana http://ugspace.ug.edu.gh 4.2.4 .2 Nutrient profile of yellow flesh kokonte Table 5 shows Total carotenoids, iron and zinc contents of kokonte. Moisture content ranged from 11.30±0.15% (sample 07) to 13.79±0.46% (sample 05). Dry matter content ranged between 86.21±0.46% (sample 05) and 88.56±0.28% for sample 01. Iron content ranged from 101.45±0.64 to 116.30±0.14 mg/100g (sample 01 and 07) and for zinc, 0.15±0.07 to 0.70±0.01 mg/100g (sample 07 and 03). Total carotenoids were not detectable (ND) in all kokonte samples. There were significant differences among all samples (p<0.05) with respect to dry matter, total carotenoids, iron and zinc. Table 5: Total carotenoids, iron and zinc contents of yellow flesh kokonte (d.m.b) Sample Moisture Dry matter Total Iron Zinc content (%) (%) carotenoids (mg/100g) (mg/100g) (µg/g ) b a c c 01 11.44±0.28 88.56±0.28 ND* 101.45±0.64 0.30±0.01 b a b a 03 11.71±0.145 88.29±0.15 ND* 113.35±0.21 0.70±0.01 a b b c 05 13.80±0.46 86.21±0.46 ND* 112.10±0.42 0.50±0.01 b a a b 07 11.53±0.11 88.46±0.11 ND* 116.30±0.14 0.15±0.07 b a a c 15 11.30±0.152 88.70±0.15 ND* 116.40 ±0.00 0.15±0.07 P- 0.00 0.00 0.00 0.00 0.00 value *ND means non detectable, Means with different superscript on the same column are significantly different at P ≤ 0.05. 4.2.4.3 Total carotenoids, iron and zinc contents of boiled yellow flesh cassava roots Dry matter content was between 23.20±0.60% (sample 05) and 30.58±0.81% (sample 07). Moisture content ranged between 69.43±0.81% (sample 07) and 76.80±0.60% (sample 05) (Table 6). Total carotenoids were between 1.22±0.05 µg/g (sample 05) and 2.14±0.11µg/g (sample 15). Iron content for the samples ranged from 118.75±0.64 to 181.85±2.05 mg/100g 45 University of Ghana http://ugspace.ug.edu.gh (sample 07 and 15) and for zinc, 0.60±0.00 to 1.30±0.00 (sample 07 and 03). There were significant differences among all samples for each parameter measured (p<0.05). 46 University of Ghana http://ugspace.ug.edu.gh Table 6: Total carotenoids, iron and zinc contents of boiled yellow flesh cassava roots (d.m.b) Sample Moisture content Dry matter Total Zinc Iron (%) (%) carotenoids (mg/100g) (mg/100g) (µg/g ) a a 01 71.60±1.09b 28.40±1.09 2.12±0.163 a c 0.65±0.07 143.40±0.57 b a b 70.90±1.09 29.10±1.09 1.35±0.05 ab b03 1.30±0.01 156.10±1.13 a b b 05 76.80±0.60 23.20±0.60 1.22±0.05 bc d 1.15±0.21 125.50±0.28 b a a 69.43±0.81 30.57±0.81 2.07±0.05 c e07 0.60±0.01 118.75±0.64 b a a 15 72.59±0.52 27.41±0.52 2.14±0.11 c a 0.80±0.01 181.85±2.05 P- 0.00 0.00 0.00 0.00 0.00 value *ND means non detectable, Means with different superscript on the same column are significantly different at P ≤ 0.05. 4.2.4.4 Total carotenoids, iron and zinc contents of boiled yellow flesh cassava leaves Samples recorded moisture content of between 72.00±2.97% (sample 15) and 77.80±0.28% (sample 03) (Table 6). Dry matter content ranged between 28.00±2.97% (sample 15) and 22.200±0.28% (sample 03). Total carotenoids were between 524.39±9.89 µg/g (sample 07) and 1323.5±15.6µg/g (sample 03). Iron values were between 95.90±0.01 mg/100g for sample 05 and 148.75±2.76 mg/100g for sample 15. Zinc values 0.60±0.01mg/100g for sample 07 and 1.30±0.01mg/100g for sample 03 respectively. There were significant differences among all samples (p<0.05). 47 University of Ghana http://ugspace.ug.edu.gh Table 7: Total carotenoids, iron and zinc content of boiled yellow flesh cassava leaves (d.m.b) Sample Moisture Dry matter Total carotenoids Iron Zinc content (%) (µg/g ) (mg/100g) (mg/100g) (%) a a c 01 72.40±1.27 27.60±1.27 633.23±3.62 d c 14.80± 0.14 0.65±0.07 a a a 03 77.80±0.28 22.20±0.28 1323.50±15.60 b c 148.25±0.07 1.30±0.01 a a c 05 75.55±0.35 24.85±0.35 662.40±44.10 c ab 95.90±0.01 1.15±0.21 a a d 07 72.95±1.06 27.05±1.06 524.39±9.89 a a 113.10±1.13 0.60±0.01 a a b 15 72.00±2.97 28.00 ±2.97 1194.79±10.01 a bc 148.75±2.76 0.80±0.01 P- 0.00 0.00 0.00 0.00 0.00 value Means with different superscript on the same column are significantly different at P ≤ 0.05. 4.2.5 β -carotene contents in selected yellow cassava roots and leaves and their products Table 8 shows the β-carotene content in selected yellow cassava genotypes and their products. β-carotene content in the fresh roots ranged between 6.74µg/100g for sample 05 and 343.99 µg/100g for sample 01. For boiled roots, beta carotene content ranged between 14.88µg/100g for sample 05 and 104.99µg/100g for sample 07. Gari recorded a β-carotene content of 0.10µg/100g for sample 7 and 3.02µg/100g for sample 03. Fresh leaves recorded β-carotene content between non detectable (ND) for sample 03 and 1911.91 µg/100g for sample 07. Boiled leaves recorded a content of between 95.60 µg/100g for sample 15 and 1875.15 µg/100g for sample 05. 48 University of Ghana http://ugspace.ug.edu.gh Table 8: Beta carotene in selected fresh yellow cassava and their products (d.m.b) Sample Fresh Boiled Gari Kokonte Fresh Boiled cassava Cassava (µg/100g) (µg/100g) cassava cassava roots roots leaves leaves (µg/100g) (µg/100g) (µg/100g) (µg/100g) 01 343.99 79.05 0.54 *ND 691.86 12.78 03 72.26 40.13 3.02 *ND *ND 417.85 05 6.74 14.88 ND 3.94 1911.91 1875.15 07 20.24 104.99 0.10 *ND 1213.78 1190.45 15 160.04 21.42 1.65 *ND 97.47 95.60 *ND means non detectable, 4.2.6 Total antioxidant activity of yellow flesh cassava products Total antioxidant activity for boiled roots, kokonte and gari were not detectable. For boiled leaves, values ranged between not detectable and 97.52±0.47%. There was significant difference among all samples (p<0.05) (Table 9). The antioxidant activity was also expressed in Alpha Tocopherol units. For boiled leaves, antioxidant activity ranged from 1.17±0.22 to 6.94±0.05 Alpha Tocopherol units. There was significant difference among all samples (p<0.05) (Table 10). 49 University of Ghana http://ugspace.ug.edu.gh Table 3: Total antioxidant activity (%) of yellow flesh cassava products Sample Boiled roots Boiled leaves Kokonte Gari a 01 *ND 90.36 ±0.10 *ND *ND b 03 *ND 96.03 ±0.72 *ND *ND c 05 *ND 0.00±0.01 *ND *ND d 07 *ND 97.51±0.45 *ND *ND e 15 *ND 85.26±0.03 *ND *ND P-value 0.00 0.00 0.00 *ND means non detectable, Means with different superscript on the same column are significantly different at P ≤ 0.05. Table 4: Total antioxidant activity in yellow cassava products in alpha -tocopherol units Sample Boiled leaves Boiled roots Kokonte Gari b 01 4.53±0.05 *ND *ND *ND c 03 1.86±0.34 *ND *ND *ND e 05 *ND *ND *ND *ND d 07 1.17±0.22 *ND *ND *ND a 15 6.94±0.05 *ND *ND *ND P-value 0.00 0.00 0.00 0.00 *ND means non detectable, Means with different superscript on the same column are significantly different at P ≤ 0.05. 4.30 Total carotenoid retention in yellow cassava products after processing Table 11 shows carotenoid retention in yellow cassava products after processing. Gari had retention between 33.88±1.73% and 61.15±0.01% for sample 05 and 07 respectively. Carotenoid retention in boiled roots was between 23.47±1.32% (sample 03) and 50 University of Ghana http://ugspace.ug.edu.gh 43.89±0.01% (sample 07). Boiled leaves showed retention of between 19.79±0.16% and 153.52±2.02%. There was significant difference among all samples (p<0.05). Table 5: Total carotenoid retention (%) in yellow cassava products after processing Sample code Boiled roots Gari Kokonte Boiled leaves 01 26.96±1.79 39.08±0.43 *ND 28.47±0.20 03 23.48±1.32 39.85±0.82 *ND 153.52±2.02 05 21.44±1.09 33.88 ±1.73 *ND 32.56±2.30 07 43.89±0.01 61.15±0.01 *ND 19.79±0.16 15 27.77±1.55 35.17±0.19 *ND 105.44±0.70 *ND means non detectable, Means with different superscript on the same column are significantly different at P ≤ 0.05. 4.3.1 Effect of processing and variety on total carotenoids, iron and zinc contents of yellow flesh cassava Cassava processing had a significant effect on dry matter and the total carotenoids (p<0.05) but had no significant effect on iron and zinc contents (p<0.25) as presented in (Table 12). The type of cultivar on the other hand (Table 13), had a significant effect (p<0.05) on the dry matter, iron and zinc contents but had no significant effect on the total carotenoids (p>0.05). 51 University of Ghana http://ugspace.ug.edu.gh Table 6: Statistical effect of processing on total carotenoids, iron and zinc contents of yellow flesh cassava Sample Mean total Mean dry matter Mean iron Mean carotenoids(µg/g ) (%) (mg/100g) zinc (mg/100g) c c a a Fresh 6.40 37.52 114.18 0.78 Roots c a a a Gari 6.20 96.26 145.12 0.66 c b a Kokonte 0.00 88.05 111.92a 0.36 c d a a Boiled 1.80 27.70 104.20 0.90 Roots b d a a Boiled 867.70 26.18 104.20 0.90 Leaves a cd a a Fresh 1780.80 33.99 159.52 9.36 Leaves P-Value 0.01 0.01 0.25 0.25 Means with different superscript on the same column are significantly different at P ≤ 0.05. Table 7: Statistical effect of cultivar on total carotenoids, iron and zinc contents of yellow flesh cassava Cultivar Mean total Mean dry Mean iron Mean zinc carotenoids (%) matter (%) (mg/100g) (mg/100g) a b b a 01 442.90 53.14 88.8 1.53 a b a a 03 338.80 54.11 148.24 2.18 a b ab a 05 416.80 51.88 114.87 2.99 a b ab a 07 491.00 53.58 125.63 1.09 a a a a 15 361.30 62.52 141.16 1.86 P-value 0.74 0.03 0.00 0.00 Means with different superscript on the same column are significantly different at P ≤ 0.05. 52 University of Ghana http://ugspace.ug.edu.gh 4.3.1.1 Effects of processing and cultivar on antioxidant activity of yellow flesh cassava Processing and cultivar had a significant effect on the antioxidant activity of yellow flesh cassava (p-value 0.0001) (Table 14 and Table 15). Table 8: Statistical effects of processing on antioxidant activity of yellow flesh cassava Treatment Mean total antioxidant activity (%) c Fresh roots 16.37 d Gari 0.00 d Kokonte 0.00 d Boiled roots 0.00 b Boiled leaves 37.88 a Fresh leaves 73.83 P-value 0.01 Means with different superscript on the same column are significantly different at P ≤ 0.05. Table 9: Statistical effect of cultivar on antioxidant activity of yellow flesh cassava Cultivar Mean total antioxidant activity (%) a 01 43.99 b 03 21.27 c 05 4.93 b 07 26.09 b 15 23.79 P-value 0.01 Means with different superscript on the same column are significantly different at P ≤ 0.05. 53 University of Ghana http://ugspace.ug.edu.gh 4.3.2 Contribution of yellow flesh cassava to RDA of vitamin A for under-5 children. The percentage of recommended daily allowance (RDA) met for vitamin A, per 100 grams of fresh yellow cassava for children under 5 years was between 1.39% (sample 05) and 70.95% for sample 01. For boiled roots, percent RDA was between 3.07% and 21.66% for samples 05 and 07 respectively. Kokonte had an RDA of between ND to 0.81%, fresh leaves reported values between 20.11% and 394.39% for samples 15 and 05 respectively. Boiled leaves also had values between 2.64% for sample 01 and 245.56% for sample 05 (Figure 1). 100% 90% 80% Cultivar 70% 15 60% 7 50% 5 40% 3 30% 1 20% 10% 0% Fresh roots Boiled roots Gari Kokonte Fresh leaves Boiled leaves Yellow flesh cassava products Figure 1: Contribution of yellow flesh cassava to recommended daily allowance (RDA) of vitamin A for children under-5. 54 Recommended daily allowance University of Ghana http://ugspace.ug.edu.gh TM 4.3.3 Comparison of icheck carotene and spectrophotometric method for carotenoid determination The spectrophotometric method for the determination of total carotenoids was compared to TM 2 the icheck carotene method for fresh roots and gari. Regression (R ) of 96.49% and 95.31% were obtained (Figures 2 and 3). 9 8 y = 1.4744x - 1.0952 R² = 0.9531 7 6 5 4 3 2 1 0 0 1 2 3 4 5 6 7 Icheck readings (mg/L) TM Figure 2: Relationship between iCheck carotene and spectrophotometric method (Gari) 55 spectrophometeric readings(mg/L) University of Ghana http://ugspace.ug.edu.gh 4 3 y = 6.5116x - 6.9852 R² = 0.9668 2 1 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 -1 -2 -3 Icheck readings(mg/L) TM Figure 3: Relationship between iCheck carotene and spectrophotometric method (Fresh roots) 4.3.4 In vitro bio-accessibility of carotenoids in yellow flesh cassava Table 16 shows the in vitro bio-accessibility of boiled yellow cassava roots, gari and boiled yellow cassava leaves. In vitro bio-accessibility of carotenoids for boiled roots ranged between not detectable to 104.42±0.88%, and for gari, values ranged between not detectable (ND) to 57.22±9.01%. For boiled leaves, values ranged between not detectable (ND) to 0.28±0.01%. There was significant difference between all samples (P<0.000). Table 10: In -vitro bio-accessibility (%) of carotenoids in yellow flesh cassava Sample Boiled roots Gari Boiled leaves b c a 01 21.49±3.53 ND 0.28±0.01 c b d 03 4.10±1.42 29.25±0.49 0.04±0.01 c a e 05 ND 57.22±9.01 ND c c b 07 ND 0.617±0.01 0.13±0.01 a c c 15 104.42±0.88 ND 0.08±0.01 P-Value 0.00 0.00 0.00 *ND means non detectable, Means with different superscript on the same column are significantly different at P ≤ 0.05. 56 spectrophotometric readings(mg/L University of Ghana http://ugspace.ug.edu.gh 4.3.4.1 Effect of processing and cultivar on the in vitro bio-accessibility of carotenoids Processing had a significant effect on the in- vitro bio-accessibility of the carotenoids, with a p- value of 0.0299. The cultivar however, did not have a significant effect on the in vitro bio- accessibility of carotenoids present in the yellow flesh cassava genotypes (Tables 17 and 18). Table 11: Effect of processing on the in vitro bio accessibility of carotenoids Sample Mean (%) in vitro bio-accessibility a Gari 26.000 c Boiled roots 17.416 b Boiled leaves 0.104 P-Value 0.03 Means that do not share a letter are significantly different Table 12: Effect of cultivar on the in vitro bio-accessibility of carotenoids Sample Mean % in vitro bio-accessibility b 01 7.25 ab 03 11.13 ab 05 19.07 b 07 0.25 a 15 34.83 P-Value 0.07 Means that do not share a letter are significantly different. 57 University of Ghana http://ugspace.ug.edu.gh 4.3. 5 Survey on consumption patterns of cassava This section of the results describes the consumption patterns of cassava and then probed into the attitudes towards adoption of yellow flesh cassava in the Ghanaian diets. 4.3.5.1 Socio-demographic characteristics of study population The socio-demographic characteristics of respondents are presented in Table 19. Two hundred and eighty seven (287) respondents were interviewed for the survey, 135(47%) being males and 152(53%) females. . 58 University of Ghana http://ugspace.ug.edu.gh Table 13: Socio-demographic characteristics of study population (N=287) Variable n (%) Age(years) 18-39 262(94) 40-59 19 (4) Above 60 6(1.9) Sex Male 135(47) Female 152(53) Marital status Single 224(78) Married/Cohabitating 50(17.4) Divorced 8(2.8) Widow 5(1.7) Educational level No formal education 8 (2.8) Primary/JHS/ middle school 53(18.5) SHS/secondary 30(10.5) Post-secondary 33(11.5) Tertiary 163(56.8) Occupation Student 131(45.6) Fixed income 64(22.3) * Non-fixed income 77(26.2) Average monthly income(GHC) 0-100 104(36.2) 100-1000 143(49.8) 1000-2000 14(4.9 2000 and above 26(9.1) Number of households with children under 15 years Yes 170(59.2) No 117(40.8) Residential status own house 57(19.9) family house 105(36.6) rented house 104(36.2) company/mission house 2(0.7) government house 12(4.2) caretaker 4(1.4) squatter 3(1.0) *Non fixed income - (beautician, carpentry, caterer, Craftsmanship, driver, hairdressing, marketer, self -employed, sound engineer, steel bender, petty trading, farming, dressmaking) 59 University of Ghana http://ugspace.ug.edu.gh 4.3.5.2 Consumption patterns of white cassava Cassava products consumption patterns data indicated that gari had the highest frequency of consumption, followed by other cassava products, cassava dough, kokonte, boiled cassava and lastly boiled leaves (Figure 4). 400 350 Cassava products 84.2 300 Other cassava products 77.5 66.7 250 63.2 Cassava dough 28.6 33.3 200 31.6 18.4 50 Boiled leaves 150 47.5 52.6 16.7 Boiled roots 100 51.1 47.4 50 Kokonte 50 79.4 78.9 83.3 Gari 0 18-39 40-59 60 and above Age of participants Figure 4: Consumption frequency of cassava products NB: other cassava products (fufu, fried cassava balls and chips, tapioca, yakeyake) 60 Percentage University of Ghana http://ugspace.ug.edu.gh 4.3.5.3 Reasons for cassava consumption among Ghanaians Reasons given for the consumption of cassava included it being a staple food, affordable, available and having lots of uses. Other reasons stated were that cassava could be used as a source of nutrients and easy to prepare. The frequencies of responses are shown in Figure 5. 40 35 33.8 28.9 30 25 23.7 20 15 13.3 10 8.5 5 0 other reasons availability affordable lots of uses staple Reasons for cassava consumption Figure 5: Reasons for cassava consumption 4.3.5.4 Knowledge of nutritive value of white and yellow flesh cassava The survey also showed that 70.4% of respondents knew the nutritive value of white cassava. Out of this population, 63.9% knew white cassava to be a major source of macronutrients, but only 6.5% of this same population knew it to be a source of micronutrients. 36.6% of the respondents had knowledge about yellow flesh cassava and 13% of this population had knowledge of the nutritive value of the yellow flesh cassava as it being a source of carbohydrates and vitamin A(Figure 6). 61 percentage of participants University of Ghana http://ugspace.ug.edu.gh 70 63.4 60 50 36.6 40 30 20 10 0 Yes No Knowledge of yellow cassava Figure 6: Knowledge of yellow flesh cassava 4.3.5.5 Acceptability of yellow flesh cassava Of the total population, 51.2% were willing to accept yellow flesh cassava. The reasons given for “willingness to accept‟ were diet diversity and curiosity. Unwillingness to accept yellow flesh cassava was due to lack of knowledge about the yellow cassava (Figure 7). 62 Percentage University of Ghana http://ugspace.ug.edu.gh 51.5 51.2 51 50.5 50 49.5 48.8 49 48.5 48 47.5 willing to accept not willing to accept Willingness to accept Figure 7: Acceptability of yellow flesh cassava 4.3.5.6 Perceived usage of yellow flesh cassava Some of the respondents (64.3%) perceived that yellow flesh cassava could be used for gari, cassava dough, starch and other cassava products that white cassava is being used for. The remaining 35.7% did not know what yellow flesh cassava could be used for (Figure 5). 63 Percentage University of Ghana http://ugspace.ug.edu.gh 70 64.3 60 50 40 35.7 30 20 10 0 don't know for some cassava porducts Perceived usage : Figure 8: Perceived usage of yellow flesh cassava 4.3.5.7 Predictors of knowledge and willingness to accept yellow flesh cassava Results of the logistic regression (Table 20) indicate that compared to people who were between 18-39 years (the reference category), those who were aged between 40 to above 59 were 4 times more likely to have knowledge about yellow flesh cassava. Married people were 2 times more likely to „willingly accept‟ yellow flesh cassava. 64 Percentage University of Ghana http://ugspace.ug.edu.gh Table 14: Predictors of Knowledge and ‘willingness to accept’ Yellow Flesh Cassava (N=284) Variable Knowledge of yellow flesh cassava Willingness to O.R(C.I) accept yellow cassava O.R(C.I) Age 18-39 1.00 1.00 18-39 versus 40-59 4.44(1.63- 12.07)* 2.18(0.81-5.92) 18-39 versus above 60 4.09(0.74-22.80) 0.50(0.09-2.80) Gender Male 1.00 1.00 Male versus female 0.72(0.45-1.12) 0.65(0.41-1.04) Marital Status Single 1.00 1.00 Single versus Married 1.81(0.95-3.47) 2.49(1.26-4.94)* Single versus Cohabitating 1.38(0.23-8.44) 3.38(0.67-17.09) Single versus Divorced 2.07(0.50-8.51) 0.281(0.03-2.56) Single versus Widow 3.10(0.51-18.98) 0.75(0.12-4.58) Educational status No formal education 1.00 1.00 No formal education versus 2.98(0.64-13.88) 1.94(0.43-8.70) up to middle school No formal education versus 1.11(0.22-5.54) 1.50(0.31-7.19) secondary No formal education versus 0.56(0.11-2.86) 0.52(0.11-2.51) Post-secondary No formal education versus 0.65(0.15-2.82) 0.90(0.21-3.70) Tertiary Ref.: Reference category * means p -value<0.05. 65 University of Ghana http://ugspace.ug.edu.gh CHAPTER 5 5.0 DISCUSSION 5.1 Nutrient profiles of yellow flesh cassava roots and leaves Moisture content is an important factor in the storage of cassava as well as in its products; very high levels greater than 12% are reported and this allows for microbial growth (Padonou et al., 2010). Low levels are therefore more favourable because it gives relatively longer shelf life (Harris and Koomson, 2011). Fresh yellow cassava roots had moisture content between (67.053±1.09) and (82.123 ±0.76) % and dry matter content of (17.877±0.760) and (32.947±1.09) %. The United States Department of Agriculture, (2009) reported 45.9 to 85.3% for moisture content and (29.8 - 39.3%) for dry matter content for yellow flesh cassava roots, (Zvinavashe et al., 2011) reported moisture content for cassava roots to be between 60.3% to 87.1%. Chávez, et al., (2008) and (Teye, et al., 2011) also recorded values ranging from 29.8% to 40.7%, with an average of 34.5% for dry matter content. Moisture content of between 57.5 and 54.8 % for yellow flesh cassava roots was reported by (Ceballos et al., 2013) and 69.1±4.17% was reported by (Maziya-Dixon and Dixon, 2015). Moisture content of the leaves was between 56.06±0.33% and 81.62±0.52% was reported by (Wobeto et al., 2006). Moisture content of 64.8 to 88.6% was also reported by (USDA, 2009). This trend from the various studies generally indicates that yellow flesh cassava is very susceptible to post harvest deterioration and therefore needs to be processed immediately after harvesting. However, (Morante et al., 2010) reported that the presence of the beta carotene trait which has an oxidative nature influences reduction of the deterioration of harvested cassava roots. 66 University of Ghana http://ugspace.ug.edu.gh Total carotenoids ranged from 792.93±0.98 µg/g and 2649.20±29.10 µg/g for fresh leaves and between 4.73±0.11 µg/g and 10.11±0.18 µg/g for fresh roots. This is in agreement with reports by (Vimala et al., 2008) who reported a total of 3.6-6.4μg/g (fresh weight.) carotenoids for yellow flesh cassava roots. Iglesias et al., (1997) also reported 7.7 to 46.9 mg/g (dry matter basis) for yellow flesh cassava roots similar to results by (Omodamiro et al.,2012) and (Esuma et al., 2012). Total carotenoid content differences could be as a result of factors such as variety, stage of maturity, geographic site of production, part of the plant which is used, conditions during planting, post-harvest handling, processing, and storage conditions (Rodriguez-Amaya and Kimura, 2004) . Iron and zinc contents in yellow flesh cassava were reported by (Wobeto et al., 2006) to be between 20.29–22.5 mg/100g and 4.20 -5.16 mg/100g. (Umuhozariho et al., 2014) also reported 5.9 to 7.62 mg/100g for zinc and iron of 21.5- 27.8 mg/100g. These reported values from literature are lower than the results from the study with iron (87.35±3.18 to 146.25±1.20 mg/100g) and higher for (zinc) 0.30±0.00 to 1.55±0.07 mg/100g for fresh yellow cassava roots. Iron (23.0-27.8 mg/100g) and zinc contents (6.4-7.6 mg/100g) were reported in fresh cassava leaves (Umuhozariho et al., 2014) and (Balal et al., 2013) reported iron contents of 7.4mg/100g for fresh white cassava leaves. Cassava roots with provitamin A carotenoids according to (Graham and Rosser 2000) and (Hess et al., 2005) have a mutually stimulating effect on iron and zinc and this could have accounted for the higher iron and zinc contents in the study. Fresh leaves on the other hand, contained 118.35±0.07 and 182.05±0.07 mg/100g (iron) and 3.75±0.64 and 15.50±0.14 mg/100g (zinc). The pH of the soil where the cassava was planted also has a high impact on Fe and Zn contents in the roots (Bortey-Sam et al., 2005). 67 University of Ghana http://ugspace.ug.edu.gh 5.2 Effect of processing on total carotenoids, iron and zinc contents of yellow cassava Cassava must be modified into different products in order to promote a good shelf life, improve transportation and marketing, decrease cyanide content and improve acceptability (Nyirenda et al., 2011). Cassava products include gari, kokonte, cassava dough, fufu among others. Cassava processing methods involves a number of operations which are done at various phases. Such activities are taking of the peels, cutting into chips or slices, mashing, milling into finer particle sizes, and grating among others (FAO, 2002). Carotenoids are highly unsaturated and therefore are more susceptible to isomerization and be oxidization (Rodriguez-Amaya, 2002). This occurs when carotenoids are exposed to heat, acids and light (Maziya-Dixon et al., 2015).Carotenoid loss happens during processing through peeling, isomerization, and enzymatic or non-enzymatic oxidation (Rodriguez- Amaya et al., 1997). Gari is moisture free, brittle but tender, creamy-white, grainy and fermented food made from cassava (James et al., 2012). It is the most popular form of processed cassava presently eaten in West Africa (Ukenye et al., 2013). Drying causes a reduction in moisture, volume and cyanide content of roots, thereby prolonging product shelf life (Westby, 2002). Osunde and Fadeyibi (2011) reported that the moisture content of gari for safe storage should be below 12.7% and their findings are in agreement with the results which reported moisture content of 2.88±0.09 to 5.25±0.07%. Total carotenoid from the study was similar to findings from (Maziya-Dixon et al., 2015) for gari who reported total carotenoid content of 3.11-15.9 µg/g. Iron content for the boiled leaves samples was not significantly different from that of the fresh leaves and was not affected much by the processing. Fermentation, a process used in making gari, is also one of the oldest and most important traditional food processing and preservation techniques (Cardoso et al., 2005). Fermentation is reported by (Achinewhu et al., 1998) to 68 University of Ghana http://ugspace.ug.edu.gh enhance the nutrient content of foods through the biosynthesis of vitamins, fiber digestibility as well as enhancing micronutrient bioavailability. This could have accounted for the high carotenoid content in the yellow cassava gari as well as iron and zinc contents. The observed increase in total carotenoid concentration after processing into gari according to (Rodriguez- Amaya 2004) could be due to the fact that carotenoids in processed foods can be obtained more easily than with those in uncooked form where the provitamin A are shielded and/or in combination with other food compounds that hinder solvent entry and extraction. The higher amounts may also be as a result of unaccounted losses of moisture and solids which would increase the retention and the total carotenoid per unit weight of the processed food (Maziya- Dixon et al., 2015). Boiling has been implicated in losses of certain micronutrient in food including iron, copper and zinc (Ahenkora et al., 1996). Boiled roots has lower total carotenoid concentration than that in gari even though some studies have reported that boiling retains more carotenoids than the roasting (Iglesias et al.,1997; Chavez et al., 2005). This could be due to the fact the wet heat treatments are more destructive on nutrients than dry heat treatment. Boiled leaves also had a reduction in total carotenoids however; the location of carotenoids in the leaves makes it more protected from heat as compared to the roots (Coleman et al., 2010). Drying in a solar dryer can appreciably reduce losses of carotenoids by protecting the food from direct sunlight. However this depends on the length of the drying period. Longer periods of drying in the solar dryer after 5 days degrade the carotenoids instead of being protective (Rodriguez-Amaya and Kimura, 2004). Vimala et al., (2011) also reported that the drastic reduction of carotenoids in this process may be due to the destructive effect of the sunlight on the stability of carotenoids. Kokonte used in the study was solar dried during the rainy season 69 University of Ghana http://ugspace.ug.edu.gh and this elongated the drying period to 7 days. This could have accounted for the degradation of the carotenoids making it non detectable. The cultivar or genotype of cassava also had an effect on the total carotenoid content after processing (Iglesias et al., 1997). 5.3 Factors affecting carotenoid retention Nutrient retention is defined as the measure of the amount of nutrient left in the processed food in relation to the nutrient originally present in the raw food (USDA, 2009). Smolin et al., (2003) reports that the time, method and temperature for the cooking are some factors that affect nutrient retention. The retention of carotenoids also decreased with a longer processing time and after a higher processing temperature, also when food is blended (Rodriguez- Amaya 2002). Gari had the highest retention of the carotenoids for the products made from the roots. This means that the carotenoid loss during gari processing was much lower in yellow gari. Boiled roots on the other hand retained the lower amount of carotenoids, whiles kokonte retained no carotenoids at all after processing. Boiled leaves retained the highest amounts of carotenoids after processing. Carotenoids in green leafy vegetables are located in the chloroplasts and also bound to protein complexes which protect the carotenoid thus giving room for more retention (Canene-Adams and Erdman, 2009). Carotenoids in the roots however are within membranes of large proteins which are crystalline in nature. These membranes are easily broken on exposure to heat making it more liable for the carotenoids to be lost (Serrano et al., 2005). Retention varies between 10% for cassava products processed to a greater extent to 87% for boiling (Maziya-Dixon et al., 2000; Thakkar et al., 2009).This was in agreement with findings from the study. 70 University of Ghana http://ugspace.ug.edu.gh 5.4 In vitro bio-accessibility of carotenoids in yellow cassava Processing methods using heat is said to enhance bio-accessibility of beta carotene by unbinding the matrix, thus facilitating its assimilation (Veda et al., 2006). Carotenoids that are present in green leafy vegetables are located in the chloroplasts and also form bonds to protein complexes which are not easily digestible. On the other hand carotenoids in the roots are within membranes of large proteins which are crystalline in nature making it more available (Canene-Adams and Erdman, 2009). This could explain why cassava roots products were more bio-accessible than the leaves. Inhibitors such as gels, cellulose fiber inhibit absorption by sustaining bile salts and preventing micelle formation which will aid at the point of absorption (Serrano et al., 2005). In vitro bio- accessibility of the carotenoids was comparable to studies reported by Failla et al., (2009). 5.5 Contribution of yellow flesh cassava to RDA of vitamin A under- 5 children Boiled roots contributed between 4 and 21% of the RDA for children less than 5 years and the boiled leaves contributed between 2.6 to 386% of the RDA for every 100 grams of food consumed which showed more potential to meeting the RDA. Gari and kokonte however provided less than 1% of the RDA for every 100grams of the cassava and as such a greater amount of the food must be consumed in order to meet a more significant amount of the RDA. Notwithstanding a positive outcome of some contribution to RDA implies that processors should put some viable measures to help reduce carotenoid losses in these products especially in gari because it is the most frequently consumed cassava food consumed. 5.6 Effect of processing and cultivar on antioxidant activity of carotenoids Processing methods such as dehydration, blanching, and canning also have an effect on the antioxidant property of the carotenoids of some dietary plants (Van Het Hof et al., 2000). 71 University of Ghana http://ugspace.ug.edu.gh Vegetables, such as carrots, mushrooms, peppers, potatoes, sweet potatoes, cabbage and tomatoes had their antioxidant activity increased when these vegetables were boiled or steamed (Halvorsen et al., 2006). Antioxidant activity of the cassava leaves also showed similar trends of increasing after boiling. However, antioxidant activity of the roots was destroyed after processing. 5.7 Comparison of icheck and spectrophotometric method for carotenoid determination Results from the study that showed the icheck method for carotenoid determination was 2 highly comparable to the spectrophotometric method with regression (R ) of 95.3% and 96.68% respectively. This was comparable to regression results reported by (Islam and Schweigert 2015) that compared the two methods using carotenoids from egg yolk. This comparison was done to validate the accuracy of total carotenoid content in this study because a quite recent method of carotenoid analysis was used in this study. 5.8 Socio-demographics characteristics Majority of the study participants were between the ages of 18-39 years and this population represents the highly reproductive part of the population who are also vulnerable to vitamin A deficiency (WHO, 2001). Majority of the participants had up to tertiary education. However their level of education did not predict corresponding level of knowledge about yellow flesh cassava. Again a majority of the population had children less than 15 years in their households; another vulnerable group to vitamin A deficiency (West,2002) and thus suggests an early adaptation to the use of yellow cassava would be a good step in curbing the prevalence of vitamin A deficiency. 72 University of Ghana http://ugspace.ug.edu.gh 5.9 Consumption patterns of white cassava Participants between the ages of 18-39 years consumed gari the most and boiled leaves the least. Gari has a longer shelf life and thus may be a factor to it being the most consumed food for that population. Onyemauwa (2010) also reported gari to be the highest consumed cassava products making it a more viable vehicle for the introduction of yellow cassava to the Ghanaian populace. However, for the participants between the ages of 40 -59 years, the most consumed cassava foods were other cassava products which included fufu. Fufu is known to be more energy dense because of the addition of cocoyam, plantain or yam and this age group preferred more energy dense foods probably because of their family size and economic status. 5.10 Acceptability of yellow flesh cassava Nutrition education is a vital tool in communicating the nutritional and health benefits of bio-fortified crops and also an important factor that affects acceptability of bio-fortified crops (Tanumihardjo, 2008). Chowdhury et al., (2011) reported that mothers in Uganda easily adopted bio-fortified foods after receiving nutrition education. From the present study, majority of the consumers who took part of the survey were not willing to accept yellow flesh cassava because they did not have any knowledge about it and this is similar to studies on the acceptability of bio-fortified foods. Consumer preferences for bio-fortified foods have also been influenced by providing nutritional information in Ghana (Groote et al., 2010). When given the same information on willingness-to-pay for orange sweet potato, orange maize, and yellow cassava, research showed that consumers liked the organoleptic traits of the bio- fortified crops and were more likely to pay more money for high provitamin A genotypes than for white genotypes (Oparinde et al., 2012,Chowdhury et al., 2011; Meenakshi et al., 2012). 73 University of Ghana http://ugspace.ug.edu.gh 5.11 Determinants of knowledge and willingness to accept yellow flesh cassava Socio-economic characteristics of poor consumers such as their income, amount of land owned, age, education level, household size and number of young minors they have in their household might play a vital role to the success of a bio-fortification program (Etumnu, 2016). Results from the study showed that socio-demographic characteristics such as age and marriage affected a person‟s knowledge and willingness to accept yellow cassava significantly. 5.12 Limitations of the study  Although iron and zinc values were very high, it‟s bioavailability was not estimated and thus was not compared to the recommended daily allowances (RDA).  RDA for vitamin A could not be computed after in vitro bio-accessibility of carotenoids because of unavailability of an HPLC to quantify beta- carotenes at that time of the study.  Micellization efficiency was not computed for in vitro bio-accessibility model for this study thus the bioavailability could not be calculated. 74 University of Ghana http://ugspace.ug.edu.gh CHAPTER 6 6.0 CONCLUSION AND RECOMMENDATIONS 6.1 Conclusions This study has established the carotenoid, iron and zinc contents of the new yellow flesh cassava genotypes grown in Ghana. 1. Fermentation, drying, roasting and boiling retained some carotenoids after processing but solar drying over a long period of time completely degraded carotenoids in yellow cassava. 2. Cassava leaves had higher retention of carotenoids but cassava roots were more bio- accessible. 3. Carotenoids in yellow flesh cassava had antioxidant properties thus had the potential to help combat free radicals in the body. 4. The antioxidant activity in the yellow flesh cassava roots were destroyed after processing into gari, kokonte and boiled roots but was higher in leaves after boiling. 5. Yellow cassava would provide a viable source of provitamin A. 6. The knowledge and “willingness to accept” yellow cassava was low among Ghanaians in the Greater Accra region. 75 University of Ghana http://ugspace.ug.edu.gh 6.2 Recommendations On the basis of the findings of this study, the following recommendations are given: 1. Further work mainly human studies or feeding trials should be done to assess the bio-availability of carotenoids in yellow flesh cassava roots and leaves. 2. Due to the significant levels of iron and zinc recorded in the studies, its bio- availability to the body should be studied and also its contribution to the RDA. 3. RDA for vitamin A should be computed after in vitro bio-accessibility of carotenoids to give a better estimate of its contribution to the RDA. 4. Micellization efficiency was not computed for in vitro bio-accessibility model for this study thus the bioavailability could not be calculated. 5. Nutrition educators should educate mothers, caregivers as well as the general public on the nutritional benefits of consuming yellow flesh cassava thus increasing its awareness and willingness to consume and accept. 76 University of Ghana http://ugspace.ug.edu.gh REFERENCES Achinewhu S.C., Barber L.I., Ijeoma I.O. (1998). 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Journal of Dairy Science 86(11): pp 3405-3415. 95 University of Ghana http://ugspace.ug.edu.gh APPENDICES APPENDIX I Recommended Daily Allowances for Iron and Zinc Age Group Recommended Daily Intake Recommended Daily Iron(mg/d) Intakes Zinc (mg/d) Infants(0−6 months) 0.27 2 Infants(7−12 months) 11 3 Children(1−3 years) 7 3 Children (4−8 y) 10 5 Males(9−13 y) 8 8 Males (14−18 y) 11 11 Males( 19−30 y) 8 11 Males (31-50 y) 8 11 Males (50-70 y) 8 11 Males (> 70 y) 8 11 Females(9−13 y) 15 8 Females(14−18 y) 18 9 Females(19−30 y) 8 8 Females(31-50 y) 8 8 Females (50-70 y) 27 8 Females(> 70 y) 27 8 Pregnancy( ≤ 18 y) 27 12 Pregnancy(19-30y) 27 11 Pregnancy(31-50 y) 27 11 Lactation(≤ 18 y) 10 34 Lactation(19-30y) 9 40 Lactation(31−50 y) 9 30 Source: Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc (2001) 96 University of Ghana http://ugspace.ug.edu.gh APPENDIX II Table 22 : RDA (vitamin A) met by 100g of yellow flesh cassava for children under 5 years Sample Fresh Boiled Gari(%) Kokonte(%) Fresh Boiled roots(%) roots (%) leaves leaves (%) (%) 01 70.95858 16.30651 0.111392 ND 142.7175 2.63627 03 14.90586 8.278054 0.622968 ND ND 86.19449 05 1.390334 3.06946 ND 0.812747 394.3906 386.8077 07 4.175126 21.65744 0.020628 ND 250.3797 245.5672 15 33.0132 4.418538 0.340364 ND 20.1062 19.72046 97 University of Ghana http://ugspace.ug.edu.gh APPENDIX III Genotype codes and their original name Sample code Original name 01 I082264 02 I011412 03 I083774 05 I011797 06 I070539 07 I083594 08 I090151 09 I083724 10 I070557 11 I082461 12 I070593 13 I090090 14 I061635 15 I083703 98 University of Ghana http://ugspace.ug.edu.gh APPENDIX IV UNIVERSITY OF GHANA Official Use only OFFICE OF RESEARCH, INNOVATION AND DEVELOPMENT Protocol number Ethics Committee for Basic and Applied Science (ECBAS) PROTOCOL CONSENT FORM Section A- BACKGROUND INFORMATION Title of Study: // Consumption Patterns, Perceptions and Total carotenoids,iron and zinc contents of Yellow Flesh Cassava Principal Elizabeth Afriyie Duah Investigator: Certified ECBASS 003/15-16 Protocol Number Section B– CONSENT TO PARTICIPATE IN RESEARCH General Information about Research The purpose of the study is to assess the consumption patterns of the regular cassava and the perception of yellow flesh cassava among consumers of cassava. Benefits/Risk of the study There is no anticipated discomfort for those contributing to this study. There is no promise that you will receive any benefit from taking part in this study. Confidentiality Your records will be kept confidential and will not be released without your consent except as required by law. Codes will be used to identify the participants instead of their names. Compensation There will no incentives provided for the participants. Withdrawal from Study 99 University of Ghana http://ugspace.ug.edu.gh Your decision to take part in this research study is entirely voluntary. You may refuse to take part in or you may withdraw from the study at any time without penalty. Contact for Additional Information If you have any questions about the research now or during the study, please contact me on 0246779814 OR Prof. Matilda Steiner- Asiedu on 0541260704 Section C- VOLUNTEER AGREEMENT "I have read or have had someone read all of the above, asked questions, received answers regarding participation in this study, and am willing to give consent for me, I will not have waived any of my rights by signing this consent form. Upon signing this consent form, I will receive a copy for my personal records." ________________________________________________ Name of Volunteer ____________________________________________ _______________________ Signature or mark of volunteer Date If volunteers cannot read the form themselves, a witness must sign here: I was present while the benefits, risks and procedures were read to the volunteer. All questions were answered and the volunteer has agreed to take part in the research. _________________________________________________ Name of witness ________________________________________________ _______________________ Signature of witness Date 100 University of Ghana http://ugspace.ug.edu.gh I certify that the nature and purpose, the potential benefits, and possible risks associated with participating in this research have been explained to the above individual. __________________________________________________ Name of Person who obtained Consent ___________________________________________ ______________________ Signature of Person who obtained Consent Date 101 University of Ghana http://ugspace.ug.edu.gh APPENDIX V Other sociodemographic characteristics Variables n(%) Frequency of white cassava consumption daily 38(13.2) twice a week 57(19.9) three or more times a week 46(16.0) monthly 28(9.8) occasionally 118(41.1) Consumption of cassava at breakfast Yes 40(13.9) No 247(86.1) Consumption of cassava at lunch Yes 143(49.8) No 143(49.8) Consumption of cassava as supper Yes 185(64.5) No 102(35.5) Consumption of cassava as snack Yes 28(9.8) No 259(90.2) Persons who consume cassava most in the household husband 33(11.5) wife 38(13.2) children 78(27.2) visitors 17(5.9) other(extended family) 121(42.2) Knowledge of nutritive value of cassava Yes 202(70.4) No 85(29.6) Sources of information on yellow cassava Radio and television 12(4.2) Family and friends 50(17.4) Other(market, farm, school, public gathering) 32(11.1) 102 University of Ghana http://ugspace.ug.edu.gh APPENDIX VI UNIVERSITY OF GHANA DEPARTMENT OF NUTRITION AND FOOD SCIENCE PERCEPTIONS AND CONSUMPTION PATTERNS OF YELLOW FLESH CASSAVA AMONG GHANAIAN CONSUMERS REF NO: ECBAS 003/15-16 Background Information 1. Please state your current age in years.................. 2. Please indicate your Sex. 1. Male [ ] 2. Female [ ] 3. What is your marital status? 1. Single [ ] 2. Married [ ] 3. Divorced [ ] 4. Widow/widower [ ] 4. What is your educational level? 1. No formal education [ ] 2.Primary/JSS/Middle [ ] 3.SSS/Secondary [ ] 4.Post-secondary [ ] 5.Tertiary and above[ ] 5. What is your main occupation? 1. Petty trading [ ] 2.Farming [ ] 3. Dressmaking [ ] 4.Student [ ] 5.Fixed salary based job [ ] 6.Unemployed [ ] 7. Other [ ] please specify ……………………………………… 6. What is your residential status? 1. Own house [ ] 2.Family house [ ] 3.Rented house [ ] 4.Company/mission house [ ] 5.Government house [ ] 6. Caretaker [ ] 7.Squatter [ ] 7. How many persons are in your house hold? ...................................................... 8. How many children <15years are there in your household? …………….. 9. What is your monthly average income (GHC)? 103 University of Ghana http://ugspace.ug.edu.gh 1. 0-100 [ ] 2. 100- 500 [ ] 3. 500-1000 [ ] 4. 1000 -2000 [ ] 5. Above 2000[ ] Consumption and Usage of Cassava 10. How often do you eat cassava? 1. Daily [ ] 2. Twice a week [ ] 3. Three or more times a week [ ] 4. Monthly [ ] 5. Occasionally [ ] 6. Not at all [ ] 11. Which cassava product do you consume and how often do you do that? Daily Twice a Three or Monthly Occasionally Not week more times at in a week all Gari Konkonte Cassava dough Cassava leaves Boiled cassava Other [ specify] 104 University of Ghana http://ugspace.ug.edu.gh 12. At what meal do you consume any of the above products? 1. Breakfast [ ] 2. Lunch [ ] 3. Supper [ ] 4. Snack [ ] 13. Who consumes most of the cassava products in the household? 1. Husband [ ] 2. Wife [ ] 3. Children [ ] 4. Visitors [ ] 5. Other [ ], specify ……………………………………………………………………………………….. 15. Why do you consume cassava? 1. Affordable [ ] 2. Have a lot of uses [ ] 3. Found everywhere and every time [ ] 4. It is a staple in my house [ ] 5.Other [ ], specify ………………………………. …………………………………………………………………………………… 16. Do you know the nutritive value of cassava? 1. Yes [ ] 2. No [ ] 17. If yes, state the main nutrients you think/know are present in cassava. ………………………………………………………………………………… 18. Do you think there is an increase in the number of people eating cassava? 1. Yes [ ] 2. No [ ] 19. If yes, what do you think is the reason for the increase in consumption? ....................................................................................................................................................... ....................................................................................................................................................... ....................................... 105 University of Ghana http://ugspace.ug.edu.gh Knowledge about yellow flesh cassava 20. Do you know/ OR have you heard about yellow flesh cassava? 1. Yes [ ] 2. No [ ] 21. . If yes, how did you know/hear about it? 1. Radio and television [ ] 2.Family [ ] 3.Friends [ ] 4.Other, please specify…………………………………………………………………………… 22. If given the option of using yellow flesh cassava, are you willing to use it? 1. Yes [ ] 2. No [ ] 23. If yes why? ................................................................................................................................................. ........... 24. If No, why? ………………………………………………………………………………………… ………………………………………………………………………………………… ………………………. 25. What uses (products) do you think the yellow flesh cassava can be put to? Please list ........................................................................................................................................... ........................................................................................................................................... ........................................ 26. Do you know of any perceived benefits for using yellow cassava? 1.Yes[ ] 2.No[ ] 27. If yes, please list ……………………………………………………………………………… 106 University of Ghana http://ugspace.ug.edu.gh THANK YOU 107