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Abstract ISSA, JOSEPH YOHANE. In Vitro Calcium Bioaccessibility in Moringa oleifera Vegetable Leaves: Potential Plant Food to Increase Dietary Calcium Intake inDeveloping Countries. (Under the direction of Dr. Jonathan C Allen). Low calcium intake, poor calcium absorption, exessive calcium losses, or some combination of these factors contribute to calcium deficiency diseases. Calcium deficiency can lead osteoporosis, reduced bone mass, hypertension and colon cancer among other diseases.Calcium in bones plays a role in bone mineralization. In other body tissues it plays roles in mediating vascular contraction and vasodilation, muscle contraction, nerve transmission, and glandular secretion.The best food sources of calcium include milk and dairy products, especially cheese and yogurt, and selected seafoods, such as salmon and sardines (with bones), clams, and oysters. Bioavailability and bioaccessibility of calcium is high in milk and dairy products due to the presence of lactose and other factors. However, calcium bioavailability or bioaccessibility is low in most vegetable plants due to presence of other substances like oxalic acid, phytate and other competing minerals. Moringa oleifera, a tropical and sub-tropical vegetable plant, has a big potential to contibute a substantive portion of the recommended daily allowance (RDA) for calcium and reduce calcium deficiency diseases. The objective of this study was to determine the calcium content, digestibility and bioaccessibility in the leaves of Moringa oleifera. Calcium content was analyzed in Moringa oleifera leaf powder, sweet potato leaves and spinach leaves using an atomic absorption spectrophotometer (AAS). This was followed by an in-vitro digestion and absorption process to determine calcium digestibility and bioaccessibility. The digestible and bioacessible calcium was measured using AAS after an in-vitro digestion and an in-vitro absorption processes respectively.The results indicated that Moringa oleifera had higher percentage of calcium than spinach and sweet potato leaves. The mean percentage calcium was 1.67±0.0014, 1.44±0.0014 and 1.55±0.00 for Moringa oleifera samples from India (M3), from Malawi (M4) and another sample from Malawi (M5) respectively. The mean percentage calcium in spinach and sweet potato leaves were 0.99±0.0007, and 1.06± 0.0014 respectively.The mean percentage digested calcium was 21.68±2.65, 40.48±3.42 and 37.24±1.66 for Moringa oleifera samples from India (M3), from Malawi (M4) and the second Moringa oleifera sample from Malawi (M5) respectively. The mean percentage digested calcium in spinach and sweet potato leaves were 1.63±0.08, and 3.01± 0.68 respectively. The mean percentage dialyzed or bioaccessible calcium was 6.82±1.80, 10.75±0.96 and 11.15 ±2.88 for Moringa oleifera samples from India (M3), from Malawi (M4) and the second Moringa oleifera sample from Malawi (M5) respectively. The mean percentage dialyzed calcium in spinach and sweet potato leaves were 0.55±0.37, and 3.08±2.05 respectively. The results showed that there was a statistically significant different in percentage calcium content from the Moringa oleifera samples from India and the two samples from Malawi. The digested calcium in the two Moringa oleifera samples from Malawi was found to be statistically similar; however both samples from Malawi were statistically different from Moringa oleifera sample from India. Calcium dialyzed was found to be not significantly different for the three Moringa oleifera samples; two samples from Malawi and one sample from India by using Tukey’s Studentized Range Test with a α=0.05. Thus percentage dialyzed calcium was higher in Moringa oleifera leaves than other vegetable leaves of spinach and sweet potato. This implies that Moringa oleifera leaves are rich sources of calcium and the calcium in Moringa oleifera can easily be absorbed into our bodies as compared to sweet potato and spinach leaves. In Vitro Calcium Bioaccessibility in Moringa oleifera Vegetable Leaves: Potential Plant Food to Increase Dietary Calcium Intake in Developing Countries by Joseph Yohane Issa A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Food Science Raleigh, North Carolina 2012 APPROVED BY: ________________________ ___________________________ G.Keith Harris, Ph.D. Rick Brandenburg, Ph.D. ________________________________ Jonathan C. Allen, Ph.D. Chair of Advisory Committee DEDICATION I dedicate this to my family and friends. ii BIOGRAPHY Joseph Issa was born on 5th of January 1982 in Blantyre, Malawi. He grew up in Malawi and did his secondary school education at Chiradzulu secondary school in the same country. He pursued his bachelor’s degree in Education Science at Chancellor College, a constituent college of the University of Malawi. Joseph graduated with a Bachelor’s degree in May 2006 with a major in Chemistry and minor in Biology. After graduating from the University of Malawi, Joseph worked in the Ministry of Education, Malawi where he worked as a secondary school teacher for the sciences. He worked as a teacher from June 2006 to January 2007. Thereafter he joined the Malawi Industrial Research and Technology Development Centre (MIRTDC) in February 2007. At MIRTDC, Joseph worked as a Chemical scientist. While at MIRTDC, Joseph got a scholarship from the United States Agency for International Development (USAID) to study for a Masters degree in July 2009. Joseph pursued a Master of Science degree in Food Science at North Carolina State University under the guidance of Dr. Jonathan Allen. iii ACKNOWLEDGEMENTS I thank all my Lecturers, for the support and encouragement given to me during my study at North Carolina State University. Many thanks should go to my Advisor and committee chair of my thesis research, Dr. Jonathan Allen. I thank Dr. Allen for all the support, encouragement and guidance that he gave me during my thesis research work. Thanks should also go to all my committee members, Dr Rick Brandenburg and Dr Keith Harris for their guidance and mentorship. Thanks to Ruth Watkins, the laboratory manager of the Nutrition Technical Services Lab, Food, Bioprocesing and Nutrition Sciences Department at NC State University and Mr. Weiting Cai for their assistance and guidance in conducting my laboratory analysis. I would like to profoundly thank my parents, relatives and friends for their moral support and encouragement during my study. Special thanks should go to the United States Agency for International Development (USAID) for offering me the scholarship that enabled me to accomplish the postgraduate studies in Food Science. I would also like to thank the management team of MIRTDC for allowing me to go for the postgraduate studies and for its support. But above all, all praise and honors should go to God for this gift of life. iv TABLE OF CONTENTS LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .......................................................................................................... viii CHAPTER 1-LITERATURE REVIEW Introduction .......................................................................................................................... 1 Calcium sources and functions ........................................................................................... 1 Calcium bioavailability and absorption ............................................................................... 5 Effects of dietary components on calcium absorption ........................................................... 6 Calcium and vitamin D ..................................................................................................... 8 Effects of calcium deficiency ............................................................................................. 10 Nutritional needs of calcium .............................................................................................. 11 Calcium toxicity ................................................................................................................. 12 Techniques for measuring calcium bioavailability .............................................................. 13 In-vivo methods ................................................................................................................ 13 In-vitro methods ................................................................................................................ 15 References .......................................................................................................................... 16 BIOACCESSBILITY OF CALCIUM IN MORINGA OLEIFERA LEAVES Introduction ........................................................................................................................ 23 History and use of Moringa oleifera .................................................................................. 23 Moringa oleifera nutrition .................................................................................................. 25 Materials and Method ...................................................................................................... 26 Sample collection and preparation ..................................................................................... 26 Sample collection .............................................................................................................. 26 Sample preparation
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