PROBING THE NUTRITIVE AND THERAPEUTIC POTENTIAL OF PAKISTANI SESAME CULTIVARS FOR SCHOOL GOING CHILDREN
By
Sabiha Abbas 2006-ag-1393 M.Sc. (Hons.) Food Technology
A thesis submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
IN
FOOD TECHNOLOGY
NATIONAL INSTITUTE OF FOOD SCIENCE & TECHNOLOGY FACULTY OF FOOD, NUTRITION AND HOME SCIENCES UNIVERSITY OF AGRICULTURE FAISALABAD PAKISTAN 2019
i ii iii iv
v DEDICATION
I dedicate this humble effort to
HOLY PROPHET HAZRAT MUHAMMAD
(PEACE BE UPON HIM)
And
My Father, Mother (Late), sisters and brothers, who never stop believing in me
vi ACKNOWLEDGEMENTS
I am proud to think that what words do justice to express my thanks to ALMIGHT ALLAH (The Omnipotent, The Omniscent, The Most Merciful and The Most Beneficient) who is the entire source of all knowleddge and wisdom to mankind. His bounteous blessing that flourished my thoughts and fulfilled my ambitions and my modest efforts in the form of this write up and made this material contribution towards the deep ocean of scientific knowledge already existing and which is a permanent source of benefit for His humanity and creatures. All praises be to the HOLY PROPHET MUHAMMAD (Peace be Upon Him), the city of knowledge, the illuminating torch and the rescuer of humanity from giving astray, Who’s teachings enlightened my heart and flourished my thoughts.
The work presented in this manuscript was impossible to be accomplished without the sympathetic attitude, utmost care, observant pursuit, scholarly criticism, cheering perspective and enlightened supervision of my sweet teacher Dr. Mian Kamran Sharif (Assiociate Professor), National Institute of Food Science and Technology, University of Agriculture, Faisalabad. I am grateful to his ever-inspiring guidance and constructive suggestions throughout the course of this effort.
I am thankful to the member of my supervisory committee, Prof. Dr. Masood Sadiq Butt, Dean, Faculty of Food, Nutrition and Home Sciences, University of Agriculture, Faisalabad, Prof. Dr. Nuzhat Huma, Director Geneal, National Institue of Food Science and Technology, University of Agriculture, Faisalabad Dr. Muhammad Shahid (Associate Professor), Department of Biochemistry, University of Agriculture, Faisalabad, for their consistent guidance to accomplish this study.
Words are lacking to express my humble obligation to my “Sweet Father, Mother (Late), Sisters and Brothers” who always longed for my successful and happy life. Their endless efforts and best wishes sustained me at all stages of my life and encouraged me for achieving high ideas of life and their hands always remain raised in prayer for my success. O ALLAH! Shower your countless blessing on them. It’s my ever pray that Allah bless them with long, happy and healthy life.
Extreme love, utmost sincerity and caring behavior of my senior fellows Dr. Faiz ul Hassan Shah, my junior fellows Mr. Ghulam Murtaza, Mr. Wahab Ali Khan and my sweet sister and friends Ms.Shamaila Abbas, Ms.Sana Sadaat, Ms. Farah Ahmad, Ms. Mehwish Liaquat. The sacrifices, they offered for me, will always shine in my mind like twinkling stars. O ALLAH! Bless them with all the happiness of life and highest status in the next coming permanent world.
If pearls were words and flowers feelings, it would be easier to express my heart felt gratitude and indebtedness to all the PEOPLES and FRIENDS who HELPED me in any way throughout my life. May Allah Almighty infuse me with the energy to fulfill their noble inspiration and expectations and further modify my competence. May Allah bless us all with long, happy and peaceful lives. Sabiha Abbas
vii
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4.5 Biological Evaluation of Sesame Flour Supplemented 107 Doughnuts 4.6 Efficacy Studies in school aged children 113 5 Summary 160 Recommendations and future directions 165 Limitations of the study 166 Literature Cited 167 Annexures 188
ix List of Tables
Sr. No. Contents Page # 3.1 Formulations used in study (g/100g) 40 3.2 Experimental diets 44 Mean squares for proximate composition of sesame flours of 4.1 49 different cultivars Proximate composition (%) of sesame flours of different 4.2 50 cultivars Mean squares for mineral contents of sesame flours of different 4.3 53 cultivars Mineral contents (mg/100g) of sesame flours of different 4.4 54 cultivars Mean squares for essential amino acids in sesame flours of 4.5 57 different cultivars Mean squares for non-essential amino acids in sesame flours of 4.6 58 different cultivars Essential amino acids composition (g/100g) of sesame flours of 4.7 59 different cultivars Non-essential amino acids composition (g/100g) of sesame 4.8 61 flours of different cultivars Mean squares for antioxidant potential of sesame flours of 4.9 64 different cultivars 4.10 Antioxidant potential of sesame flours of different cultivars 65 Mean squares for bioactive components in sesame flours of 4.11 68 different cultivars Bioactive components (ppm) in sesame flours of different 4.12 69 cultivars Mean squares for sensory evaluation of white and black sesame 4.13 71 flour supplemented doughnuts Effect of white and black sesame flour supplementation on the 4.14 72 color of doughnuts Effect of white and black sesame flour supplementation on the 4.15 74 aroma of doughnuts Effect of white and black sesame flour supplementation on the 4.16 75 taste of doughnuts Effect of white and black sesame flour supplementation on the 4.17 77 texture of doughnuts Effect of white and black sesame flour supplementation on the 4.18 78 chewability of doughnuts
x
4.19 Effect of white and black sesame flour supplementation on the 80 mouthfeel of doughnuts Effect of white and black sesame flour supplementation on the 4.20 81 overall acceptability of doughnuts Mean squares for proximate composition of sesame flour 4.21 85 supplemented doughnuts Effect of treatments on the proximate composition (%) of 4.22 86 sesame flour supplemented doughnuts Mean squares for mineral contents of sesame flour 4.23 89 supplemented doughnuts Effect of treatments on the mineral contents (mg/100g) of 4.24 90 sesame flour supplemented doughnuts Mean squares for texture analysis of sesame flour supplemented 4.25 93 doughnuts Effect of treatments on the textural characteristics of sesame 4.26 95 flour supplemented doughnuts Mean squares for color characteristics and calorific value of 4.27 97 sesame flour supplemented doughnuts Effect of treatments on the color and calorific value of sesame 4.28 98 flour supplemented doughnuts Mean squares for the water activity, peroxide value, 4.29 thiobarbituric acid number and growth of sesame flour 100 supplemented doughnuts Effect of treatments and storage on the water activity of sesame 4.30 101 flour supplemented doughnuts Effect of treatments and storage on peroxide value (meq/Kg) of 4.31 103 sesame flour supplemented doughnuts Effect of treatments and storage on thiobarbituric acid number 4.32 105 (mg/kg) of sesame flour supplemented doughnuts Effect of treatments and storage on the mold count (×102 CFU/g) 4.33 106 of sesame flour supplemented doughnuts 4.34 Mean squares for PER and NPR of experimental diets 108 Effect of experimental diets on PER and NPR in Sprague Dawly 4.35 109 rats 4.36 Mean squares for TD, BV and NPU of experimental diets 111 Effect of experimental diets on the TD, BV and NPU in Sprague 4.37 112 Dawly rats Mean daily dietary intake of study groups (n=45) through 24-h 4.38 114 recall method
xi 4.39 Mean squares for the anthropometric measurements of school 116 aged children 4.40 Effect of protein enriched sesame fortified doughnuts on height 117 of school aged children 4.41 Effect of protein enriched sesame fortified doughnuts on weight 119 of school aged children 4.42 Effect of protein enriched sesame fortified doughnuts on body 120 mass index (BMI) of school aged children 4.43 Effect of protein enriched sesame fortified doughnuts on head 122 circumference (HC) of school aged children 4.44 Effect of protein enriched sesame fortified doughnuts on mid 123 upper arm circumference (MUAC) of school aged children 4.45 Mean squares for the serum ferritin and serum zinc levels of 124 school aged children 4.46 Effect of protein enriched sesame fortified doughnuts on serum 126 ferritin of school aged children 4.47 Effect of protein enriched sesame fortified doughnuts on serum 128 zinc of school aged children 4.48 Mean squares for complete blood count (CBC) of school aged 132 children 4.49 Effect of protein enriched sesame fortified doughnuts on 133 hemoglobin (Hb) of school aged children 4.50 Effect of protein enriched sesame fortified doughnuts on red 135 blood cells (RBCs) of school aged children 4.51 Effect of protein enriched sesame fortified doughnuts on packed 138 cell volume (PCV) of school aged children 4.52 Effect of protein enriched sesame fortified doughnuts on mean 140 corpuscular volume (MCV) of school aged children 4.53 Effect of protein enriched sesame fortified doughnuts on mean 143 corpuscular hemoglobin (MCH) of school aged children 4.54 Effect of protein enriched sesame fortified doughnuts on mean 145 corpuscular hemoglobin concentration (MCHC) of school aged children
4.55 Effect of protein enriched sesame fortified doughnuts on 148 platelets and white blood cells (WBCs) of school aged children 4.56 Effect of protein enriched sesame fortified doughnuts on 149 neutrophils and lymphocytes of school aged children
xii 4.57 Effect of protein enriched sesame fortified doughnuts on 151 monocytes and eosinophils of school aged children 4.58 Mean squares for serum electrolytes of school aged children 152 4.59 Effect of protein enriched sesame fortified doughnuts on blood 153 sodium, chloride and potassium of school aged children 4.60 Mean squares for the renal function test of school aged children 155 4.61 Effect of protein enriched sesame fortified doughnuts on blood 156 urea and serum creatinine of school aged children 4.62 Mean squares for liver function test of school aged children 158 4.63 Effect of protein enriched sesame fortified doughnuts on 159 bilirubin (total, conjugated and unconjugated) of school aged children
xiii List of Figures
Sr. No. Contents Page # 4.1 Effect of varieties and treatments on overall acceptability of white 83 and black sesame flour supplemented doughnuts 4.2 Percent increase in serum ferritin of school aged children 127 4.3 Percent increase in serum zinc of school aged children 129 4.4 Percent increase in Hb of school aged children 134 4.5 Percent increase in RBC of school aged children 136 4.6 Percent increase in PCV of school aged children 139 4.7 Percent increase in MCV of school aged children 141 4.8 Percent increase in MCH of school aged 144 4.9 Percent increase in MCHC of school aged children 146
xiv List of Annexures
Sr. No. Contents Page #
I Composition of Micronutrient Premix per 100g 188 II 9-Point Hedonic Score System for Sensory Evaluation 189 III Institutional Biosafety/Bioethics Committee 190 IV Informed Consent Form 191 V Subjects Distribution for Efficacy Trial 193
xv ABSTRACT
Protein energy malnutrition (PEM) has emerged a key health problem in Pakistan. It mostly affects the health of pregnant women and school going children. Almost 1/3 children are underweight, and rest are affected by stunting and wasting. Hence, there is a great need to explore use of locally grown traditional crops into modern food products for alleviating malnutrition among the masses. The present project was designed to develop protein enriched, and micro-nutrient fortified doughnuts from different Pakistani white (TH-6, TS- 5, TS-3, Til-89) and black (S-122, S-117, S-131, Latifi) sesame cultivars. The proximate and mineral composition of sesame flours of white and black cultivars revealed varying degree of moisture, protein, crude fat, crude fiber, ash and NFE. Regarding antioxidant potential and bioactive components, total phenolics, sesamin, sesamol and sesamolin were ranged from 1.56±0.025 to 7.32±0.219 mg GAE/g, 1521.00±27.30 to 3996.00±129.90 ppm, 3121.00±86.60 to 4303.00±133.37 ppm and 1532.33±31.89 to 3564.00±65.82ppm, respectively. Among the different cultivars, the maximum value of sesamin was observed in TH-6 whereas sesamol and sesamolin concentrations were higher in S-122. It is obvious from the results that sesamin and sesamol contents were higher in white sesame cultivars whereas sesamolin was more in black cultivars. Sensory evaluation of doughnuts revealed maximum liking for product made from TH-6 (8.02±0.24) due to better appearance, palatability, fine texture, chewiness, mouthfeel, and overall acceptability. Concerning various levels of supplementation, doughnuts having 10 g/100g (7.67±0.24) to 20 g/100g (7.43±0.18) of either white or black sesame flours were more suitable due to delicious nutty aroma & flavor of sesame, mild brownish appearance, uniform texture, nutty mouthfeel and overall impression of the end product. Based on consumer acceptability and better- quality attributes, TH-6 (white sesame cultivar) with two supplementation levels (10g/100g and 20g/100g of sesame flour) was selected for further development of doughnuts and assessed for nutritional contents, storage stability, calorific value, protein quality and feeding trial to see the effect of these products on nutritional and serum biochemical profile of school aged children. Biological evaluation of selected formulations showed the highest PER (0.86±0.17), NPU (2.81±0.25), true digestibility (64.51±0.84%), biological value (69.64±1.08%) and net protein utilization (61.32±0.96%) in rats fed with diet containing (20g/100g) sesame supplemented doughnuts. After biological evaluation, selected treatments of doughnuts alongwith control were served to school children to observe their effect on nutritional biomarkers and anthropometric measurements through the analysis of blood and urine. Maximum weight gain (1Kg) was detected in the children served with 20g/100g of sesame supplemented doughnuts. It is inferred from the current intervention, that the addition of sesame flour in snacks may improve the nutritional quality of these products and ultimately the consumers. Moreover, the consumption of doughnuts containing 10 to 20g/100 sesame flour may deliver 140% additional protein, as well as % daily values of Zn and Fe. Such products are ideal candidates for school nutrition programs especially in developing countries to reduce the severity of malnutrition.
xvi CHAPTER 1
INTRODUCTION
The protein energy malnutrition and micronutrient deficiencies are escalating in individuals from under-developed and developing economies mainly due to poverty and food insecurity. The children are severely affected by stunting, wasting, under-weight, Marasmus and Kwashiorkor due to consumption of monotonous diets and reliance on poor sources of protein. Consequently, edema, wasting of body tissues, subcutaneous and muscular fat, poor health and inadequate mental and physical activities are significant among the children (Shakeel et al., 2009). In Pakistan, nutrition surveys and food balance sheets have shown existence of varying degrees of nutrient deficiencies just because of low quantity as well as quality of protein especially vulnerable groups of the population (GOP, 2011). School aged children are more prone towards wasting and stunting. These circumstances demand dietary mediations to reduce the deficits and develop their health status. Globally, malnutrition is affecting all age groups; however, the vulnerable segments of the society and poor masses in developing countries are being severely challenged. South Asian countries are facing this problem especially Pakistan, Bangladesh and India; where almost half of the world’s children and most of the mothers are undernourished (UNICEF, 2013). Pakistan is ranked at 11th among 119 countries based on three prime indicators i.e. prevalence of child mortality, child malnutrition, frequency as well as ratio of the protein deficient individuals. Current statistics reveals about 870 million undernourished people worldwide. Furthermore, about 852 million of them are residing in under developed countries (Global Hunger Index, 2016). According to Global Hunger Index 2017, there is overall decline in the average global score for undernutrition from 29.9% in 2000 to 27% in 2017. However, South Asian (30.9%) and African countries still have serious levels of hunger due to deep inequalities and insufficient access to quality foods (Global Hunger Index, 2017). Consequently, more than half of the child demises occur due to malnourishment (Cheah et al., 2010). It causes low physical and mental development in children and subsequently poses minimum interest in educational attainments and productivity (Chirwa and Ngalawa, 2008). The poor nutritional status of mothers, meager maternal care practices, illiteracy, lack of empowerment, insufficient
1 consumption of foods, frequent infections, substandard health and care services and frequent ailments are the main contributories of malnutrition in poor economies (Linnemayr et al., 2008). Worldwide, protein energy malnutrition (PEM) hits every fourth child; the estimated number of stunted are 182 million followed by 150 million under-weight. Geographically, PEM is affecting about 70% population of Asia. In Pakistan, about 8 million children (under- 5) and 77 million women are suffering from array of nutritional deficiency disorders. According to the latest National Nutrition Survey, 1/3rd (31.5%) children are under-weight, whereas rest ones are hit by stunting (43.7%) and wasting (15.1%). Amongst the micronutrient deficiencies, 54% are vitamin A deficient, 39.2% are zinc deficient whereas 61.8% are iron deficient. Similarly, 18% of the non-pregnant females (15-49 years old) are under-weight as well as deficient in vitamin A (42%), iron (50.4%), vitamin D (47.6%) and zinc (42%), respectively (GOP, 2011). Micronutrients are indispensible for endurance and development of human beings. These are essential for the production of hormones, enzymes and other substances in the body. In food, these nutrients in balanced quantities are important to maintain optimum health along with decreasing death ratio among the children, infants and mothers. Micronutrients encompass vitamins and minerals. Vitamins are chemical compounds (organic) essential for body in small quantities for the maintenance of health. Likewise, minerals are crucial for regulation of blood pressure, balancing electrolytes and normal working of nerve impulse. Deficiencies emerging from inadequate nutrients intake are responsible for deviant physiological body functions (Bowman and Russell, 2006). Biochemical investigation of vitamins and trace elements since last 50 years has heightened the understanding of their mode of actions and reasons of essentiality in diet. The previous studies have recognized the importance of trace elements in enzyme activity either as co factors or within active site of enzymes. Several roles have also been recognized which are equally important. Increased production of reactive oxygen species is termed as Hyper-metabolism resulting mainly from oxidative metabolism and ultimately causing oxidative damage at various places in cell particularly polyunsaturated fatty acids in nucleic acid in nucleus or cell membrane. There is a well-established antioxidant mechanism in body for neutralizing harmful impacts of these oxidants, so there is vital function of micronutrients in this aspect either as intermediate in quenching these free radicals or through direct involvement. Micronutrients
2 have other roles as well like regulation of gene transcription through activation of particular gene (Evans and Halliwell, 2001). A predictable sign of micronutrients significance is disease development by severe deficiency which can be controlled by supplying these nutrients. There is a well-coordinated system for ensuring the exact concentration of each micronutrient in right place of body fluids or tissue at a specific stage of illness. Hence plasma iron, zinc and selenium decline when copper rises and vitamin concentration in plasma also declines (Shenkin, 2006). Micronutrients deficiency is a main global health problem. Above 2 billion people in the world are facing deficiency of more than one micronutrient especially zinc, iodine, iron and vitamin A. The major causes of deficiency are poor access to micronutrient rich foods like vegetables, fruits, fortified foods and animal products being expensive or unavailable in local markets. Micronutrient deficiency enhances the mortality from measles, pneumonia, malaria, diarrhea and other infectious illnesses (WHO, 2009). The estimated data has shown about 53% school aged children suffering from nutrient deficiencies which have negative impact on their physical and mental development and enhance susceptibility to infectious illnesses (Allen et al., 2006). Recent studies from Philippines, Thailand, Nicaragua and Colombia have revealed 23 to 38% prevalence of anemia. Iron deficiency is also common in School age children of United States of America (Halterman et al., 2001) specifically in adolescent girls. Likewise, iodine deficiency has high prevalence (60-90%) in school children from various Eastern, Asian and African Mediterranean countries (Sebotsa et al., 2003; Abuye et al., 2007). Inadequate intake of folate and vitamin D has also been found in adolescents and children of Europe. Multiple micronutrient deficiencies occur simultaneously because of poor diet. Low dietary intake in developing countries especially foods of animal origins which are good source of protein, iron, zinc and B12, can cause severe illnesses leading to death (Longfils et al., 2005). International and local organizations are continually struggling to reduce the extent of micronutrients deficiencies (Akhtar et al., 2011). Currently, almost 20% of the worldwide population is being affected by the iron deficiency which is ranked at the top among micronutrient deficiencies (Huma et al., 2007). Moreover, anti-nutrients present in the staple foods of Asians further makes the situation worse due to reduced bioavailability (Hussain and
3 Maqsood, 2010). These deficiencies can be eradicated by fortifying staple diets, practicing dietary diversification and taming nutrition awareness among the people (Ahmad et al., 2013). Food fortification has been recognized as an effective and long-standing method to improve the levels of micronutrients among the masses (WHO, 2001; Mannar and Sankar, 2004). Widespread acceptance of fortified products is due to numerous associated benefits e.g. minimum changes in existing dietary practices, nutrients adjustment to reduce toxicity, fortification with more than one nutrient, less investment in foods and cost-effective activities for small scale producers (Cook and Reusser, 1983; Allen et al., 2006). Fortification is now being controlled by the governmental organizations in countries especially whose population is at greater risk. Nutritional disorders can be eradicated by adopting global best practices, experiences and standard dietary approaches (Lynch, 2005). Fortified foods like biscuits, corn soya blend and oil enriched with iodized salt, iron and vitamin A are frequently provided as part of food rations in emergency situations. (WFP, 2004). Sesame (Sesamum indicum L.), also named as gingely, sim-sim, till and beniseed, belong to family Pedaliaceae. Worldwide, it is ranked at 9th position among commercially grown oilseeds and is commonly cultivated in tropical and subtropical regions of Asia. The production of sesame at global level is about 3.66 million tonnes mainly shared by the Asian and African countries (Sarkis et al., 2014). China, India, Myanmar and Sudan are the major growers, collectively producing about 60% of the world's total production. The chemical composition of sesame seed revealed presence of oil (50%), protein (25%), water (5.8%), carbohydrates (10-15%), ash (4.7-5.6%) and fiber (3.2%). Moreover, it is rich source of minerals and antioxidants like vitamin E, phosphorus and calcium (Lee et al., 2005). It has balanced proportion of essential as well as non-essential amino acids, with high net protein utilization (54%) and chemical score (62%). (Alobo, 2006). These features make sesame a potential applicant for supplementation in collection of food products to increase their protein, amino acid profile and quality as well. Sesame seed is also termed as “queen of the oil seed crops” due to the presence of maximum oil content (Gandhi and Taimini, 2009). Wheat (Triticum aestivum) is the topmost staple grain of Pakistan with cultivated area of 8.7 million hectares and 25.49 million tonnes/annum yield. It is one of the major crop in the country with 1.7% share in GDP (GOP, 2018). White flour is obtained after milling and generally termed as refined flour. Fine flour include moisture (13%), crude protein (13%),
4 crude fiber (0.62%), crude fat (1.78%), ash (1.32%) and carbohydrates (70%), respectively (Abril et al., 2015). Wheat flour is mainly used for the production of chapaties (a type of unleavened bread) in Pakistan. Additionally, different baked products like biscuits, cookies, leavened pan bread and cakes etc are also prepared using refined flour. Snacks are foods consumed between meals and are regarded as an excellent mode of improving nutrient and energy requirements. According to a study, US children consume snacks on an average of six times/day. According to Canada's Food Guide 2011, the governmental departments like Health Canada are suggesting people to consume more nutritious and healthy snacks. Snacking has become a special feature of 21st century life style. Certainly, children have more attraction towards snacks like patties, samosas, fries, pakoras etc. that may comprise carcinogens because of repeated frying and heating in the similar oil. Doughnuts are sweet snacks made from a fried dough confection which are greatly relished by children in many countries. These are ring-shaped with a hole in the center and usually topped with sugar icing. These are usually consumed as such or taken with milk or coffee, at doughnut shops or fast food restaurants. Processed legumes and oilseeds have pronounced potential as snack food for school aged children (Estevez et al., 2000). Sesame flour being rich in protein, vitamins, minerals and dietary fiber can be used for the production of novel and inexpensive value-added products. The present research project was designed to assess whether sesame cake flour supplemented energy- and nutrient-dense doughnuts served to school going children have significant impact on their nutritional and biochemical profile. The goal of the study is to develop protein and micronutrient enriched doughnuts in order to alleviate nutrient descrapencies in the children. The main objectives of the study include: Characterization of Pakistani sesame cultivars for nutritive and therapeutic potential Develop and optimize micronutrient fortified sesame cake flour supplemented doughnuts Consumer acceptability of the doughnuts through sensory evaluation Nutritional and biochemical evaluation of nutrient dense doughnuts through efficacy studies CHAPTER 2 REVIEW OF LITERATURE
5 The malnutrition has direct link with economical and communal living standards of the masses. It is one of the main reasons of mortality and morbidity among the children particularly in less affluent countries. Supplementations, fortification along with awareness among masses through educational campaigns have proven productive in the past. However, food-based methodologies are regarded best to deal with such problems. In this context, snack foods are becoming more popular day by day and nutritional status of children is being improved. In the era of modern nutrition, these snacks are used as a means for delivering important nutrients. Hence, conventional snacks are now being supplemented and enriched with array of nutrients. Furthermore, modern approaches of supplementation have further make such foodstuff more appealing for children. In this study, sesame-based doughnuts have been developed especially school-aged children to improve the levels of protein and deficient micronutrients. The literature related to existing malnutrition scenario, nutritional interventions, pharmaceutical and nutraceutical benefits of sesame and importance of snacks for the maintenance of good health is presented below under the following captions: 2.1. Malnutrition: Global and Local Scenario 2.1.1. Global Situation 2.1.2. Pakistani Context 2.1.3. Hidden Hunger 2.2. Strategies to Combat Malnutrition 2.3. Sesame in Human Nutrition 2.3.1. Nutritional Facts 2.3.2. Pharmaceutical and Nutraceutical Benefits 2.3.3. Food Applications 2.4. Snacks 2.5. Conclusion
6 2.1. Malnutrition: Global and Local Scenario 2.1.1. Global situation Malnutrition is a worldwide concern, commonly among the poor and vulnerable ones distributing all age groups in developing countries (WHO, 2002). It is a physical discrepancy that rises due to improper consumption of food as a function of quality as well as quantity. According to WHO, it is a physical state resultant of complete or relative shortage of one or more essential nutrients (Black et al., 2013). Malnutrition also refers to numerous diseases, each with a particular reason associated with one or more nutrients like iron, protein and iodine. It might be the outcome of low income resources, more number of children under-5, absence or poor breastfeeding, food insecurity, illiterate mothers, poor knowledge about weaning foods and their timely introduction. Secondary malnutrition is associated with malabsorption of nutrients, metabolic disorders, infections or sometimes may be due to lack of immunity for fatal diseases. In spite of innovations and technologies due to green revolution and genetically modified organisms (GMO’s), still 3.1 million children are malnourished of age under five (Vandersmissen and Peeters, 2015). Today, there is much need of realization and taking appropriate steps to stop millions of us who are susceptible to nutritional deficiencies from becoming the victim of nutritional deficiency diseases (Victora et al., 2010). Malnutrition has emerged one of the leading health issue in South Asia particularly in Pakistan, Bangladesh and India which are the home of about half of the globally malnourished children. Malnutrition badly affects physical and mental growth in children which further leads to poor awareness and educational attainments (Chirwa and Ngalawa, 2008). Malnutrition in Asian children is associated with insufficient intake of nutritious foods, frequent infections & other ailments, poor maternal care practices & nutritional status of mothers, food insecurity and meager health & care services ((WHO, 2002; Linnemayr et al., 2008). Malnutrition is linked with high risk of infections and morbidity, which leads to death (Liu et al., 2015). Globally, protein energy malnutrition (PEM) affects every fourth child. In 2011, around 52 million children were wasted, 165 million were stunted and comparatively maximum prevalence was found in South Asia and Sub-Saharan Africa (FAO, 2015). Globally, almost 795 million people do not have adequate food to live a healthy energetic life. Most starving people reside in developing countries, where almost 12.9% population is undernourished.
7 In the developing world, approximately 66 million primary school-aged children go to school empty stomach. According to World Food Program, US$ 3.2 billion are required each year to satisfy these hungry children (WFP, 2012). Malnutrition is not only limited to children but is also widespread among the females of reproductive age. Maternal malnutrition has a negative impact on fetal growth, which causes low birth weight and elevated risks of mortality and childhood infections. Children who confronted with these health risks in early life, have a greater chance of stunting and underweight, which results in irretrievable and damaged cognitive, motor and health impairments. Most of the irreparable damage due to malnutrition arises during pregnancy period and in the first 2 years of life, hence it emphasizes the significance of negotiating in this period (Black et al., 2013). 2.1.1.1. Stunting The important factor in early period of life particularly (1-5 years) is growth and development. Insufficient intake of vital nutrients at the crucial age may results in underweight, stunting and wasting. It is defined as the low height for age, in less than two years of age due to inadequate intake of nutrients and prolonged infections. Its irreparable effects include reduced cognitive growth and poor performance in school. Almost 1/3rd of children
8 still needed in sub-Saharan Africa where more than 1/3rd of the countries are affected by the malnutrition (WHO, 2015). 2.1.1.2. Wasting It is termed as low height for weight and is considered as a strong indicator of mortality among children under the age of five years. It occurs due to severe food shortage and disease. It comes under the severe under-nutrition category (WHO, 2010). This is the most overwhelming condition leading to death. Globally, 11% decline in wasting has been noticed since 1990. The trenchant part of the world especially South Asian countries are suffering from wasting (16% of children), which means that one in six children is prone to moderate or severe wasting. The share of India is again more as compared to other countries i.e. 25 million children are affected by wasting. In Africa, nearly 1 in 10 children i.e. 9% under the age of 5 are vulnerable group of wasting. Considerable association was found in countries with maximum occurrence of wasting along with food insecurity and more frequent climatic crises which further promote spread of infectious diseases, cultural and social confines to reduce the problem of wasting. 2.1.1.3. Underweight It is a type of malnutrition which encompasses both wasting and stunting. It is described as low weight for age of the reference population (WHO, 2010). It is a composite indicator and may therefore be difficult to interpret. Globally, about 101 million children of age less than 5 years are underweight. This is equal to 16% of all the children around the globe. The condition is more terrible in South Asia 59 million children are underweight (33%) followed by 21% residing in Africa region (30 million). There has been 37% reduction in the last two eras with the highest decline (upto 87%) was achieved by the Commonwealth and Europe of Independent States followed by the Pacific and East Asia i.e. 73% 73% (De Onis et al., 2004). However, only 26% improvement was noticed in the remaining areas of the world. 2.1.1.4. Low birth weight It is the term in which infant have weight less than 2,500 grams at birth. Worldwide, more than 20 million infants fall in this category. The major contribution is from India i.e. 33% making it largest donor of this problem. 1 out of 4 newborns in developing countries has low birth weight. Countries contributing low birth weight load are Pakistan (1.05 million), India (7.05 million), Philippines (0.50 million), Nigeria (0.80 million) and Bangladesh (0.70 million), respectively. In developing countries, there is no proper documentation of children weight at
9 birth time. Thus, the actual number of children is greater in the under-developed nation when data collection was not practiced (Blossner et al., 2005). It reflects the existence of child care services in such areas. The World Health Assembly has established an aim to minimize this problem at the lowest level i.e. 30% in the next decade which looks like an excellent task under prevailing conditions (WHO, 2009). 2.1.1.5. Overweight It is explained as higher weight for height. Globally, it has emerged an important health problem and developing countries are being affected more by it. Childhood undernutrition and overweight exist in various countries that may lead to a two-fold burden in malnourishment (WHO, 2010). Historically, over-weight was frequently noted in developed countries whereas the worldwide records gathered by WHO have indicated an odd relation of overweight and countries having low to moderate income which contain almost 69% of overweight children. Still, occurrence (8%) is more in developed as compared to low income countries. Globally, about 43 million children of age less than 5 years are overweight (De Onis et al., 2010). In Africa, this figure has touched to 3% since last 2 decades. Parallel rising trend was noticed for the remaining regions throughout the world. 2.1.2. Pakistani context The population of Pakistan is 207.77 million (GOP, 2018). It comprises equal ratio of males and females. About 7% of population is above 60 years while 33% is under the age of 15 years. It has also been noticed that birth rate has increased upto 0.3% whereas death rate has reduced to 0.1% (GOP, 2015). Malnutrition is prevalent in Pakistan between all age groups and improvement not encouraging over the last 2-3 decades. In Pakistan, more than half of the mother and children are suffering from malnutrition (NNS, 2011). The country is at second number in the stunting (44%) after Afghanistan. 1/3rd of children in Pakistan are underweight. Whereas, stunting (44%), wasting (15%) and iron deficiency anemia (33%) is also widely prevalent. It has been reported that stunting prevalence differs significantly (22-76%) within Pakistani districts. The minimum values for wasting and underweight were <2.5% whereas the maximum figures for wasting and underweight were 42 & 54%, respectively (Di Cesare et al., 2015). Micronutrient deficiency is also ubiquitous at very high rate. A huge amount of children having age less than 5 are having deficiency of vitamin D (40%), vitamin A (56%), zinc
10 (37%) and iron (33 %). This situation hinders their growth and makes them susceptible to different ailments, disabilities and death. There is prominent inequity in urban and rural populations regarding nutritional indicators. 14% women of reproductive age are undernourished (BMI <18.5 kg/m2) with comparatively high percentage in rural, poor and uneducated women. Currently, 85% of Pakistani females from all age groups are deficient in vitamin A (43%), vitamin D (68.9%), zinc (47.6%) and iron (51%). When pregnant women found with scarcity of essential nutrients, they and their babies are at greater threat of long-term diseases and early death. Only iodine status has been improved in most of the provinces due to Universal Salt Iodization program being launched in 1989 under iodine deficiency disorder control program (USAID, 2013). Iron Fortification in wheat flour has been initiated to address the deficiency of iron in Pakistan. Nutrition is an essential part of defensive and corrective health care facilities at the national as well as international level. Many interventions have been developed by the collective efforts of food, health, industry, agriculture and education departments (UNICEF, 2013). Currently, there is more weightage on the improvement of infant and young child nutrition (IYCN) by beginning the breastfeeding right after 1 hour of birth, breastfeeding exclusively for half year (first 6 months), introduction of nutrient dense, enriched foods and continue breastfeeding with complementary diet until 1 year of age. All IYCN signs in Pakistan are ‘undesirable low’ as compared to other countries in the South Asia region. Pakistan is noticeable for having the lowermost rates for early initiation of breastfeeding, beginning of complementary food on right time, exclusive breastfeeding for 6 months and high ratios for bottle feeding. Recent data suggests initiation of instant breastfeeding in 18% of all births, whereas exclusive breastfeeding was performed in only 38% of infants less than 6 months. Around 20% of children with age of 2 months and 46% (9-11 months) were described using bottle feeding. Only 19% of the children aged from 4-5 months were given solid to semi-solid foods. However, the main concern is poor compliance of the dietary practices. About 15% children get least acceptable diet and 22% of them noticed with very less diet diversity (GOP, 2011; PDHS, 2013). 2.1.3. Hidden hunger Hidden hunger is a primary health problem in most of the developing nations, triggered by the absence of vital nutrients i.e. vitamins and minerals. It is the outcome of insufficient intake of
11 vitamins and minerals than that of body requirements. Actually, food is deficient in micronutrients mainly due to consumption of monotonous diets over prolonged period. The children and pregnant & lactating women are considered the most vulnerable group of micronutrient deficiency due to greater requirements for these nutrients. For a pregnant woman, there is great danger of death in childbirth or birth of mentally retarded or underweight baby. Micronutrient status of a lactating mother determines development and health of infant specifically during 6 months’ age. Micronutrient deficiency in a young child enhances risk of death because of infectious disease leading to impaired mental and physical development (WHO, 2009). Deficiency can develop frequently during emergency or can cause critical situation if already present. This occurs because food crops and livelihoods are lost, food materials are interrupted and there is break out of diarrheal disease. These results in nutrient loss and malabsorption which pave the way of more infectious diseases, suppressing appetite and enhancing micronutrients requirement to help fight diseases. In disasters or emergencies, food aid rations are designed to fulfill nutrient requirements and are distributed in sufficient amount and in regular pattern. Another way is provision of foods fortified with micronutrients (FAO, 2012). There is need to fortify such foods taking into consideration fact that other unfortified foods will meet a part of micronutrient requirements. South Asian countries like India, Pakistan, Bangladesh, and Sri Lanka have the highest ratio of malnourished children in the world. These are considered major obstacle in the growth of economy and health of the nations. Poverty is the fundamental cause of child under-nutrition. Deficiencies of iodine, iron, zinc, vitamin D, and A in these countries are responsible for multiple health problems and high death rate. Micronutrient malnutrition is widely prevalent in developing countries. Children and pregnant women of child bearing ages are more prone to such deficiencies (Akhtar et al., 2011). The deficiencies of iron, iodine and vitamin A, are prominent while vitamin D and zinc deficiency data are not available in developing regions (Ejaz and Latif, 2011). Although, many efforts have been done to decrease the victim of malnutrition, yet it affects the health and cognition of numerous children. Acute to chronic deficiencies of zinc and iron have negative impact on growth, mental and reproductive health that causes difficulties in pregnancy, which further leads to birth defects and abnormalities. Health effects linked with the lack of vitamin A includes xerophthalmia that is also called night
12 blindness and Bitot’s spots, eventually causing the growth of ulcer in cornea and keratomalacia. Deficiency of vitamin D causes rickets and osteomalacia and is known as a main risk for cephalopelvic imbalance (Talat et al., 2010). Certainly, deficiencies of micronutrient hinder the growth of productivity and economy and aggravated poverty by declining the capability of physical work, failure of mental development and promote school absents. Deficiencies of micronutrient involve trillions of dollars and in developing countries this cost is intensified where the position appears to exist for many decades (Stein and Qaim, 2007; UNICEF, 2008).
2.1.3.1. Vitamin A deficiency (VAD) It has been estimated globally that one third of children (pre-school-aged) are suffering from VAD and the most susceptible group belongs to developing nations like South Asia. Approximately, one to two million deaths in pregnant women are due to the complications in childbirths and almost 44-50% of the pre-school aged children are the victims of VAD (Kaleem, 2004; WHO, 2009). Globally, India is having the largest number of vitamin A deficient child (World Bank, 2005). About 62% pre-school children are vitamin A deficient (Laxmaiah et al., 2011). In rural areas, the diets of children were found deficient in vitamin A, with usual intake about 66 to 81% low as compared to the recommendations i.e. 400mg. a and approximately 84% of pre-school children did not get 50% of their recommended dietary allowance (NNMB, 2006). About 60% children deaths in Pakistan occur due to diarrheal and respiratory infections. These health ailments are severe in VAD individuals. Many studies showed that about 5.7 million children under the age of five are affected by VAD and among these 0.057 million are the victims of xerophthalmia (Mahmood et al., 2008). Current National Nutrition Survey has shown a massive rise in VAD (5.9% in 2001 to 30.3% in 2011) among the women in Pakistan (GOP, 2011). Among the South Asian developing economies, Sri Lanka has achieved an excellent success in health outcomes and literacy even with limited resources. On the other hand, a significant progress was not seen in the monarchy of public health and nutrition (Rajapaksa et al., 2011). Accordingly, vitamin A supplementation program was formulated to improve its status in preschoolers, primary school children, adults and lactating and pregnant mothers. In Bangladesh, 18.5% women are suffering from VAD (serum retinol < 0.70mmol/l). Similarly, school age children (11-16years) have < 0.70mmol/l serum retinol level (Ahmed et
13 al., 2012). Hence, vitamin A supplementation program was introduced, which have proven effective to control VAD in country. Poverty is considered as one of the most important indicators for severe VAD in these counties. Sugar fortification in Honduras and Guatemala is one of the success stories in order to eliminate VAD (Lee et al., 2008). 2.1.3.2. Iron deficiency anemia (IDA) Globally, Iron deficiency anemia (IDA) is at the top, hitting almost all age groups and genders with more susceptibility in preschool-aged children, adolescent girls and pregnant & lactating mothers. Lower motor and mental scores were also found in infants due to iron deficiency. This problem has significantly increased the risk of mortality and morbidity among the children. Anemia is considered a primary cause of maternal and child deaths in less developed countries (WHO, 2011). Subsequently, cognition and productivity is being affected deleteriously due to IDA (Fuglestad et al., 2012). In 16 districts of India, adolescent girls (90%), pregnant women (85%) and non-pregnant women (66%) were iron deficient mainly due to iron deficient diets and failure of menstruating and pregnant women to get additional iron supplies (Toteja et al., 2006). Likewise, IDA in Pakistan has revealed to affect 65% children under-5 and 90.5% pregnant women (Baig-Ansari et al., 2008). Moreover, vitamin
B12 and folate deficiencies are also very common (Iqbal and Kakepoto, 2009). Several reasons are found for high incidence of IDA in Pakistan which include poor hygiene, poverty, lack of sanitation, consumption of cereal-based diets and poor dietary habits. Similarly, there is strong relationship between early age pregnancies and IDA (BBS, 2005). In the presence of phytates, bioavailability of iron is disturbed, and it fails to meet up the requirements during pregnancy and growth phase, low dietary intake of iron, parasitic infections and menstrual blood loss have aggravated the risks of IDA in Bangladesh. Most established biomarker for iron deficiency is serum ferritin. Full range of iron status is evaluated through total body iron calculated from serum ferritin concentrations (Frith et al., 2000). It is the most precise biochemical test that relates with relative total body iron reservoirs. Precondition for iron deficiency is low serum ferritin level which reflects iron depletion. The optimum range of ferritin is 13.0-150ηg/mL for females and 30.0-400ηg/mL for males (Cobas, 2010). Clinically, the threshold level of 20.0ηg/mL is a positive indicator of iron deficiency (Turgeon et al., 2000; Milmani et al., 2005). 2.1.3.3. Zinc deficiency
14 Zinc deficiency is a major public health issue. Globally, it effects almost half of the total population. It is expected that 68-95% of the population in North America and 1-13% in Europe, have lower concentrations of zinc. Zinc deficiency is more prevalent in children from developing economies. It is a main risk factor in children under 5 years for mortality and morbidity causing approximately 800,000 extra mortality cases each year (Haider and Bhutta, 2009). Zinc deficiency among pre-school age children in 5 states of India was 43.8% (Kapil and Jain, 2011). Zinc deficient children are more prone to mortality linked with infections like malaria, pneumonia and diarrhea. Therefore, there is greater occurrence of foodborne illness in developing countries (Yakoob et al., 2011). Likewise, non-pregnant women of central India (52%) and adolescent girls in Delhi (49.4%) were zinc deficient (Menon et al., 2010). In Pakistan, National Nutrition Survey conducted in 2011 has depicted an alarming situation in pregnant (48.3%) and non-pregnant (41.6%) women (GOP, 2011). Zinc deficiency is also associated with poor pregnancy outcomes. Serious problems have been observed in Zn deficient lactating mothers (Lindstrom et al., 2011). Zinc deficiency is attributed to the low bioavailability of zinc available in plant-based diets and low intake of foods from animal sources (Hettiarachchi and Liyanage, 2012). Zinc supplementation strategy can be used as a preventive measure to decrease mortality and morbidity rate. Likewise, zinc supplementation also gives a positive result on linear growth (Imdad and Bhutta, 2011). Several studies showed significant evidences of zinc intervening strategies that they can decrease child deaths (30,000- 75,000) by preventing diarrhea in Bangladesh (Jones et al., 2003; Ahmed et al., 2012).
2.2. Strategies to Combat Malnutrition
In under-developed countries, commercial and suitable strategies i.e. fortification, bio- fortification, supplementation, dietary diversification and public health and nutrition education programs can be used effectively to improve micronutrient deficiencies. The success of these strategies includes the systematic understanding of foods, fortificants and their bioavailability in body (Akhtar et al., 2011). Proteins have a vital role in nutrition and is mainly important in developing countries where the protein consumption is low. Scientists are trying to figure out other protein sources for both nutritional supplements and functional food ingredients due to insufficiency and expensive cost of animal proteins (Eleazar et al., 2003). The internationally adopted strategies to tackle malnutrition are briefly described as below:
15 2.2.1. Food fortification Addition of some vital micronutrients i.e. minerals and vitamins in food products in order to enhance its nutritional quality as well as provision of health benefit at very low risks is referred as food fortification (Allen et al., 2006). This strategy is considered as the most economical among the public health interventions. In this approach, main emphasis is on selection of suitable vehicle for the delivery of nutrients to the masses. Availability of this food should be made mandatory as well as it should be easily accessible to everyone who is going to be targeted. The selected food to be fortified will give best results if there are no issues related to food security in past. After selection of suitable vehicle, proper selection of fortificants is the next step (Akhtar et al., 2011). The fortificants should have maximum bioavailability without affecting the sensory attributes of the product. After processing, the element of interest in required quantity should be present in the portion size (Allen et al., 2006). In developing countries, food fortification has resulted in the effective control of mineral (zinc, iodine and iron) and vitamin (A, D, C, B1, B2, B3 and folic acid) deficiencies. Salt iodization program was started firstly in Switzerland and United States back in 1920s (Burgi et al., 1990). Afterwards, numerous countries have adopted this strategy. In 1940s, cereals fortification was initiated when thiamin, niacin, and riboflavin were used to fortify cereal based products. In a very short time, this becomes common practice to add iron in the diets of children who were deficient in iron and having anemia. Subsequently, vitamin D in United States was used to fortify milk whereas in Denmark, margarine is fortified with vitamin A. Folic acid fortification has been implemented by more than 20 countries of Latin America, United States and Canada. Folic acid is being used for the strategic fortification of wheat flour by these countries (Arlappa et al., 2011). Recently, fortification has become attractive choice for the less developed world. Planned strategies have been developed by numerous countries at national level for its effective implementation. Zambia is the leading country where sugar fortification has been done successfully. This form of fortification could be utilized by other countries of the region as well in order to alleviate the vitamin A deficiency (Darnton-Hill and Nalubola, 2002). Although, this strategy always contributes positive results but there is scarcity of enough studies which can illustrate its effects on nutritional status of the vulnerable population. It is consistent with low socioeconomic status of the population and has direct impact on the health
16 outcomes. Success stories of the mass fortification programs can be assessed by doing efficacy trials or reports which are valuable documents for conducting such type of programs. These are done in controlled conditions so the impact can be assessed with the help of known parameter to check its effectiveness (Allen et al., 2006). Food fortification has got extreme popularity between nations facing malnourishment due to being socially acceptable, cost effective and more economical. Wheat is the staple food in South Asian developing countries therefore it has been emphasized that whole wheat flour should be fortified with multiple micronutrients in order to curtail these deficiencies among the masses. In 2007, in Pakistan flour fortification program was started and mainly focused in the province of Khyber Pakhtunkhwa where it is easily available to about 0.5 million populations (MI, 2012b). However, still the coverage remains to be 11% of the total population. According to the officials, currently 275 mills across the country are adding iron and folic acid in various types of wheat flours (GOP, 2014). 2.2.2. Bio-fortification Bio-fortification emphasizes on improving the micronutrient contents of edible parts of crops along with their bioavailability. Additionally, naturally bioavailable organic minerals are being assimilated by plants and ultimately impart natural texture and taste of the food. It is considered one of the most cost-effective approaches with the potential to mitigate malnutrition in recent years (Go´mez-Galera et al., 2010). Conventional plant breeding and biotechnology are being used in this strategy to make more nutritious staple foods in order to deliver micronutrients to the poor people of less developed world. Plant breeding programs have been employed to increase the levels of vitamins and minerals in crops having deficiency or genetic variations of these nutrients. In African and Asian countries, Harvest Plus Program pursued the conventional breeding as a main strategy for targeting the staple crops i.e. wheat, cassava, maize, rice, sweet potato, beans and pearl millet. Restricted genetic variation from sexually compatible plants in germplasm is the root cause on which these breeding strategies basically depend (Hirschi, 2009). Transgenic techniques are used as an alternative to those cases where breeding approaches cannot meet the criteria of micronutrient contribution. There are no taxonomic restrictions in genetical engineering and artificial genes can also be used. These strategies have main benefit that the investment is only needed at developmental and research phase. Subsequently, the crops that are nutritionally improved, are completely sustainable. Bio-
17 fortification is expected to be further beneficial on long-term basis than that of other interventions because it removes difficulties such as the dependence on compliance and infrastructure (Zhao and Shewry, 2011). A research study was conducted at International Rice Research Institute (IRRI) to produce rice varieties with improved zinc and iron status in 1992. With the collaboration of Department of Plant Science in University of Adelaide, Australia almost 7000 records were assessed. The samples were found to have vast differences in zinc and iron contents. Concentrations of iron were ranged from 6.03 to 24.4mg/kg whereas zinc was ranged from 15.3 to 58.4 mg/kg in 1,138 examined samples. Among the conventional varieties, “Zuchen” and “Jalmagna”, were found to have double amount of iron and 50% more zinc as compared to commonly grown varieties, IR64 and IR36. Numerous aromatic rice varieties like Basmati-370 from Pakistan & India and Azucena from the Philippines also exhibited higher zinc and iron contents (Gregorio et al., 2000). In wheat, there was four to five times more variation between the highest and lowest zinc and iron contents in grains amongst several hundred accessions (Ortiz-Monasterio and Graham, 2000). Rice varieties having high zinc and iron contents were tall and low yielding therefore not appropriate for modern agriculture. Crossbred between old and high yielding varieties have produced high levels and high yield of these micronutrients. For instance, an improved crossbred line, IR68144-3B-2-2-3, high yielding variety i.e. IR72 and tall stature old variety i.e. Zawa Bonday from India has high iron content in the grain (about 21mg/kg) in un-milled brown fraction of rice. Its production is equivalent with that of high- yielding rice varieties. A human efficacy study (9-months feeding trial) was conducted in young females of Philippines using milled rice of this variety (Haas et al., 2005). Bio-fortified feeding of the rice showed increased total dietary iron consumption (17%) and a mild increase in total body iron and serum ferritin without any change in hemoglobin. Non-anemic objects response was higher for body iron and ferritin. This study demonstrated that the intake of bio- fortified high iron rice improved the body iron by 20% (Yan et al., 2010). Research conducted at International Center for Tropical Agriculture (CIAT) revealed that different varieties of common bean had 60-80% more zinc than commercially grown ones. Advanced breeding strategies can further be utilized in order to enhance the zinc levels in these developed varieties (Beebe et al., 2000). VAD is mostly prevalent in the people who just consume cereal grains and tubers. Therefore, there is a need to improve the vitamin A status in cereal and tuber cops.
18 The main objective for pro-vitamin A bio-fortification is increased accumulation of beta- carotene and carotenoids which can be attained by guiding and increasing flux into carotenoid biosynthetic way or by down-regulating the throughput of beta-carotene (Sayre et al., 2011). The former strategy has best example of the simulation in the production of Golden Rice. Globally, bio-fortification is a propitious agricultural-based approach to improve nutritional needs of undernourished populations. So, handsome resources should be allocated for bio- fortification plans (Pray et al., 2007). 2.2.3. Supplementation In this approach, nutrients are given in relatively high doses to the target groups in significantly absorbable form for the reduction of malnutrition. Nutrition defines it as use of some sort of supplements to alleviate any nutritional deficiency which is clinically confirmed e.g. use of vitamin A for infants and multivitamins by the pregnant women. Sometimes, doses (only 2- 3/year) are sufficient to meet the nutritional requirements. The major hurdles reported by former supplementation programs are poor compliance and disorder in supplies (Mannar and Sankar, 2004). Pre-school child mortality (35%) is mainly due to the Vitamin A deficiency. Vitamin A supplementation is the most effective success story interrelated to supplementation programs for the preschool children and is required in 2 mega dosages only (UNICEF, 2004). Though, iron supplementation is more challenging than that of vitamin A as it is needed to be used on regular basis so logistic deliveries pose a serious problem in the success of this program. Supplementation of iron having side effects was a problem of some poor compliance. Resultantly, iron deficiency anemia is affecting 40-60% children in less developed countries (Malkanthi et al., 2010). Zinc supplementation is advantageous specifically where other approaches are difficult to implement. Sufficient levels of zinc are crucial for having good wellbeing. Conversely, to perform any planned program on mass level, there is requirement of assistance at country level (IZC, 2004). 2.2.4. Dietary diversification Cultural, economic and social barriers significantly bound the intake of diversified foods that usually transmit essential vitamins and minerals to efficiently lower micronutrient deficiency load between the susceptible ones (Allen et al., 2006). Essential nutrients are incorporated in the diets of target population in order to fulfill the nutritional needs. This approach is more
19 advantageous for being cost-effective, culturally acceptable and sustainable in comparison to the other strategies employed and does not demand any exterior support. The main goals focused are to increase the accessibility, food utilization and availability with a higher content and bioavailability of micronutrients around the year. It emphasizes on changes in traditional or household approaches, patterns regarding food selection and practices concerning food production for manufacturing and processing of indigenous foods. Efficient implementation of these approaches can be achieved through knowledge of the local dietary patterns used, taboos, preference, food beliefs and capability to do variation in attitudes and practices (Gibson et al., 2006). It is based on dietary guiding principles that are designed to recover micronutrient nutrition. Populations susceptible to micronutrient deficiencies, usually eat cereal grain as chief food, though these supplies sufficient amounts of energy and proteins but they contain micronutrients in poor quantities (UNICEF, 2006). Commonly, inclusion of oranges, carrots, beef, lentils and spinach in the staple foods will justify the recommended concentrations of nutrients for iron, zinc and vitamin A deficiencies (Rosalind et al., 2006). Although, food- based approaches need longer time to rise the consumptions or to improve the micronutrient status in large quantity but are reflected as more effective method. For instance, to enhance the protein status and diversify micronutrients, the mixture of cereals (beans and peanuts) and legumes are being used.
2.2.5. Nutrition education and counseling Awareness campaigns in communities can perform a significant part to lessen micronutrient deficiencies along with globally practiced approaches like fortification, dietary diversification and supplementation. Educational involvement alone has caused a significant development in iron status and nutrition knowledge in Sri Lanka (Senanayake et al., 2010). The overall nutritional level of underdeveloped countries can be strengthened by giving education to women on the importance of dietary care, weaning foods during reproductive phase and exclusive feeding practices. As mild and moderate malnutrition are not apparent, thus education of mothers about the growth of children is very important. They should be able to observe whether their children are growing normally. During reinforcement and replenishment, packages are sometimes provided for a specific period of time to direct specific guidelines. If mother is not receiving proper education on dosage, preparation and storage the plan will never be a successful outcome (Mason et al., 2006). Several countries have programs
20 in place for evaluating malnutrition in young children's. Among these programs, Integrated Management of Childhood Diseases Program, was established in 1992 with the main goal to find diseases and malnutrition in children at early stage (Armstrong et al., 2004). Furthermore, there is more emphasis on medical treatment of children. It also reveals awareness about exclusive breastfeeding, and complementary feeding of infants to reduce the incidence of diseases. 2.2.6. Monitoring and evaluation The fundamental task to check the significance any nutrition program lies in quantitatively determining how much development has been achieved? This is very essential, as it not only explains the entire condition, but also helps to identify some key findings that are suggested based on previous results. This provides food printing and motivation for others to overcome malnutrition. Unfortunately, because of its complexity, cost and fear of corruption, this has not been accomplished. Impact assessment criteria are very important. Regardless of the criteria for impact assessment, it must answer the following question: Does the nutritional contents in the end-product increased to a predetermined level? Are nutrient-rich foods met and targeted by the consumers? Does the intensive project increase the level of target nutrients in the target population? What are the impacts of intervention programs on specific groups of people, such as children, elderly and the women? What is the measurable change in micronutrient levels in the target population? Are there functional outcomes after the intervention like as morbidity, infectious diseases, impaired growth and mortality? (Health Survey, 1988; Allen et al., 2006). A variety of impact assessment methods can be used, but it is important to allocate a specific financial plan while planning an intervention because it includes expensive testing methods. In order to make the evaluation process impartial, it should be conducted under the management of an independent team. If the services of international organizations are conducted at the national level, they can be sought in this regard (Allen et al., 2006). Malnutrition is the leading problem in the Asian countries. Most parts of Ethiopia rely on rainfed agriculture, leaving the population at risk to food insecurity and drought. However, the country has managed to reduce mortality and stunting below the age of five in the past decade. Between 2000 and 2011, the mortality rate under the age of five years was assessed to have dropped from 139 deaths to 66, approaching the 36% target of Millennium Development Goal 4. The rate of stunting in children under-5 also declined during the same period, from 57 to
21 44%. However, even though Ethiopia’s GNI per capita has increased from US$ 130 in 2000 to US$ 400 in 2011, more than 5 million children are still stunted. Both the poor and the well-off families have stunted growth, showing that food insecurity and economic growth alone are not enough to decrease children's growth retardation (Rajkumar et al., 2012). In India, children (60 million) of age under-5 are malnourished. Even in Maharashtra, one of the richest states in India, 39% of children under-2 years were stunted during 2005 to 2006. However, the state’s determined actions and concerns about service delivery resulted in 16% decline just in seven years (NFHS, 2012). This improvement was attributed to better feeding methods, mother's care and overall changes in living environment. Infants and children are provided with scientifically proved dietary interventions along with the nutrition educational campaigns delivered to their mothers are used to increase their living standards, eliminate the distances and deliver quality services for their nourishment. The most vulnerable areas and sectors are given more significant influence. During each pregnancy, carefully monitoring of weight gain and special counseling and support to the mothers play important role in the reduction of low birth weight children. This happened due to strong collaboration between planning, management, monitoring and evaluation departments. Nepal, another South Asian country, has made impressive human development progress over the last decade. The proportion of the population living on 22 distribute single-use MNP pouches to families through a program called Gulazyk, which can be mixed into solid or semi-solid supplements at home. A Kyrgyz phrase refers to a rich tradition contributing nutrients and energy from soldiers or travelers. A slogan was developed: 'Gulazyk - For Children's Health and Mind'. Each child’s caregiver received 30 points. At the same time, a national maternal, infant nutrition campaign was launched to educate caregivers on their diet during pregnancy and the significance of exclusive breastfeeding and adequate complementary feeding. It was jointly developed by the government and partners including the UNICEF, the Swiss Red Cross and US Center for Disease Control and Prevention (CDC). Rural health volunteers educated the parents about primary childhood development and gave them specially developed Gulazyk branded children's book for their children. Vast media campaign for the promotion of optimal feeding and extensive monitoring resulted in successful outcomes (KMH, 2011). Sri Lanka, a middle-lower income country, has effectively improved child health and nutrition in the last two decades. The mortality rate under the age of five years dropped from 29 in every 1,000 live births to 12 and in infants the range decreased from 24 per 1,000 live births to 11 in 1990 to 2011. Progress has been made in breastfeeding; the exclusive breastfeeding rate for infants under six months has risen from 53% to 76% during 2006-2007. Latest estimates have shown that about 80% of babies receive breastfeeding within the first hour of life (UNICEF, 2009). However, in rural areas and highland tea gardens, malnutrition is higher than in other parts of the country. The approach to reduce under nutrition is to use legislature and services to support breastfeeding mothers. Sri Lanka’s attainments in escalating the rate of exclusive breastfeeding are attributed to high levels of political commitment, leading to defensive legislation, advanced health care system and favorable environment. They have dedicated group of organizations and professionals meant for promotion of breastfeeding. Prior to childbirth, full-time and well-trained on-site staff of public health midwives has contact with mothers during childbirth and after delivery. There are various strategies for creating a wide range of awareness at all levels, especially in mother support groups. It shows that even under difficult circumstances, miraculous occurrences will not happen without striving to improve people’s living conditions and provide education (WHO, 2015). 2.3. Sesame in Human Nutrition 2.3.1. Nutritional facts 23 Sesame seed belongs to the genus Sesamum of family Pedaliaceae. Nutritionally, it is considered as an important protein source because of higher amounts of some essential amino acids as compared to other seed proteins (Radha et al., 2007). Additionally, it has plenty of nutrients essential for the maintenance of optimum health. In developing countries, poverty is greatly hampering the food accessibility. Furthermore, inadequate supply of animal protein is the leading cause of certain health ailments in the populations. The scientists are striving hard to explore low cost plant protein sources for incorporation into formulations for better nutritional quality (Coulman et al., 2005). In the past, much emphasis was on oilseeds including cotton, rapeseed, soya, sunflower and peanutfor extraction and utilization of their protein isolates. Recently, researches have probed defatted sesame flour for its incorporation in food products to enhance the protein contents and curtail associated health disorders (Kanu et al., 2007). Sesame seeds are widely used to treat respiratory tract infections, infant cholera, diarrhea, diarrhea and other intestinal as well as bladder diseases. Sesame powder is also used to control amenorrhea, dysmenorrhea, ulcers and hemorrhagic acne (Alobo, 2006). Brown or black seeds are mainly used for oil extraction. The defatted sesame powder was replaced with millet flour in different proportions to prepare sesame enriched biscuits. There was significant increase in the protein contents of resultant products. As the level of sesame replacement increased, the diameter and weight of the biscuits decreased, and the thickness and spread factor were increased. The sensory evaluation results showed improvements in flavor and crispness of the biscuits. However, color of the products with higher levels of sesame flour was less appreciated by the panelists (Alobo, 2001). In another study, sesame biscuits were prepared by blending defatted sesame powder (5-8%) in a regular recipe and assessed for their microbiological and chemical properties. The results revealed increase in protein contents compared to traditional products. The sensory evaluation of end products showed their wide acceptability by the consumers (Gandhi and Tamini, 2009). The chemical composition and functional characteristics of full fat and defatted sesame seed (Sesamum indicum L.) like foam stability and capacity, oil and water uptake, bulk density, nitrogen solubility, emulsion capacity were evaluated. Defatting process results in increased amount of carbohydrates, crude protein, crude fiber, ash and mineral contents. The defatted flour has relatively good foaming capability, water absorption, constancy and the ability to 24 emulsify, but the oil absorption ability and bulk density were reduced. The solubility of nitrogen depends on the pH, the lowest pH was 4 and the highest was 8. The maximum nitrogen solubility of defatted flour (95%) and the maximum nitrogen solubility of full fat flour is 95%. The functional properties of full fat and defatted flours exhibited their utilization in making diverse foods (Egbekun and Ehieze, 1997). Sesame oil is mainly composed of unsaturated fatty acids (85%), which contain 35-43% of oleic acid, 37-47% of linoleic acid, 5-10% of stearidonic acid, 9-11% of palmitoleic acid and traces of linolenic acid (Khalida et al., 2003; Latif and Anwar, 2011). Compared with other vegetable oils, sesame oil is the most stable one to oxidative rancidity (Namiki, 2007) due to the presence of distinctive tocopherols and lignans e.g. p-hydroxyphenylpropane. Sesame and flaxseeds are abundant in lignans (water-soluble) and almost insoluble in oil, and are mainly found in sesame cake flour. Raw sesame seeds contain sesamolin and sesamin, major phenolic compounds found only in this plant (Wanasundara and Shukla, 1997). In one study, 14 Japanese cultivars were examined for the lignin content. The amount of sesamin was higher than sesamolin in white variety (0.2-0.5), and the proportion of sesamolin was more in the black variety (0.6-1.0). Other ingredients such as sesamol, pinoresinol glucosides, sesamolinol and sesamin were lower in phenols. These physiologically active ingredients have many pharmacological properties i.e. lower blood cholesterol and lipid levels, provide anti- inflammatory properties, enhance hepatic fatty acid oxidase and neuroprotective effects on brain damage or hypoxia (Moazzami et al., 2007). Many of these different bioactive compounds are now being used as food additives in the production of different functional foods e.g. nutraceuticals, pharmaceutics, cosmetic preparations and fungicides due to the antiseptic properties of sesame (Namiki, 2007). 2.3.2. Nutraceutical and pharmaceutical benefits Numerous nutraceutical and pharmaceutical benefits of sesame and its bioactive components have been reported in the literature. Lignans of sesame possess antioxidant properties and health enhancing activities. Several bioactive components i.e. sesamin and sesamolin have been found in sesame seeds (Sirato-Yasumoto et al., 2001). These have been found to increase the liver mitochondrial function and peroxisome oxidation of fatty acids in rats. Consumption of sesame seeds has been shown to increase the ratio of gamma-tocopherol of plasma and stimulating the activity of vitamin E in prevention of cancerous and cardiac diseases (Cooney 25 et al., 2001). Sesamin is quiet heat stable as obvious from its 90% retention after baking (Abe et al., 2001). Cephalin, a phospholipid in sesame seed, has been reported to have hemostatic activityranging from 133,168 to 233,856 ppm. Traditionally, fibers have been used as anti- diabetic, anti-tumor, anti-ulcer, cardioprotective, chemoprotective and laxatives. The fiber content in the sesame seeds ranged from 27,100 ppm to 67,000 ppm and contained up to 166,000 ppm in the leaves. Sesame seeds contain lecithin, which has hepatoprotective and antioxidant activity ranging from 58 ppm to 395 ppm. Lecithin may also be effective in decreasing liver steatosis in patients with longstanding parenteral nutrition and successfully treating dermatitis (Jellin et al., 2000). Anti-cancerous activity is also found in myristic acid found in sesame seeds at 328.0-1788 ppm. Sesame oil is abetted in pharmaceutical industry and is utilized as a solvent in intramuscular injections. It has nutritional, soothing and ointment characteristics and also works as a laxative. Chinese are also using this oil for the treatment of toothache and gum diseases for many centuries. It is also known that consumption of sesame oil lowers cholesterol due to high polyunsaturated fat content. Other properties include the treatments of headache, dizziness and blurred vision. Indians also use this oil as an antibacterial mouthwash for relieve in anxiety and insomnia (Annussek, 2001). Moreover, sesame oil has large amounts esters of linoleic in the form of triglycerides, which can selectively inhibit the growth of malignant melanoma (Smith and Salerno, 2001). In a study, the consumption of sesame lignans causes a reduction in Fe2+ induced oxidative stress in rats. Furthermore, rats consuming sesame oil have shown lower levels of serum glutamate pyruvate transaminase, activities of oxaloacetate transaminase and thiobarbituric acid reactive substances in liver. These enzymes levels depict safety against Fe2+ induced oxidative stress (Hemalatha et al., 2004). Several scientists have reported the presence of antioxidant and free radical lowering activities of sesamol by using radiolysis technique of nanosecond pulse (Juan et al., 2005). Sesame cake extract has also been studied for having good free radical scavenging potential of antioxidants (Suja et al., 2004). Antioxidant activity of 2, 3-epoxysesamone, hydroxysesamone and chlorosesamone toward Cladosporium fulvum was recognized in a study. After roasting, sesamin remains 90% as such of its original level due to being thermostable in nature (Hasan et al., 2001). It shows its sustainability for non-food and food applications. In another study, 80% aqueous ethanol whole white and black sesame extract and their hull fractions were 26 examined for Trolox equivalent antioxidant capacity assay, decreasing the rate of low density lipoprotein (LDL) cholesterol, free radical lowering capacity, metal chelating capacity and total phenolic content (TPC). Black sesame hull exhibited greater antioxidant activity than that of white one (Shahidi et al., 2006). 2.3.3. Food applications Sesame and its co-products have extensive usages. Intact seeds are considered as wholesome food ingredient. Its meal is being used in confectionery and bakery products instead of extracting good quality oil (Naturland, 2002). Nutty flavor of sesame makes it an excellent item in order to add texture, artistic value and taste in a lot of bakery products including bread, sesame bars, cookies, bread sticks, doughnuts, crackers, cereal mixes, cakes and buns. It has also been widely used for making a paste of milled sesame seeds known as tahini or sesame butter, which is used with crackers, pastry, rolls, breads, cakes and breadsticks like products (Nzikou et al., 2009). The seeds can also be utilized in confectionary and candies, in ground and processed forms, mixed with honey or sweet syrup, in Middle East, East Asia, and South Asia while their paste and starch are main ingredient to make a special sesame tofu known as goma-dof in Japan. These are also sprinkled over various types of desserts, margarine and salads. These are also being used for garnishing of various Mexican and East Asian Foods. Sesame seeds are also used in micro-atomized protein foods for un-weand babies (Morris, 2002). Sesame meal, obtained after extraction of oil, has exceptional nutritional attribute due to the presence of high quality protein. An outstanding balance of amino acids is present in defatted sesame flour. Sesame protein is rich in cysteine and methionine (6.1%) which are sulfur- containing amino acids; these are typically limiting amino acids in leguminous plants but have low lysine content (3.1%). Its seed cake is commonly used as a protein supplement in animal feed industries. Additionally, it is used as an excellent source of supplementation in numerous food products in order to increase the protein quality and content. Nutritional worth of food products has been effectively increased by supplementing sesame seeds along with peanut and soybean. These are also being used to increase the nutritional status in some weaning foods of infants (NAERLS, 2010). A clear white sesame seed is produced adopting a special hulling process. The resultant sesame seeds are then dried, washed twice and used on hamburger buns. White color of seed remains 27 maintained on the buns due to this special hulling and cleaning process even after baking.McDonalds, American fast food restaurant chain, purchases about 1/3rd of the imported crop from Mexico for developing sesame seeds enriched buns. They are also sprinkled over bread and then consumed in Sicily. Sesame seeds are commonly used in cakes in Greece, while they are a main ingredient of African soup known as Togo. Sesame flour is creamy, light brown, edible powder from sesame seeds. It has maximum protein content (27-32%) and sesame oil (10-12%). They have three times more calcium compared to an equivalent measure of milk (Morris, 2002). Refined sesame oil is considered as a good quality oil because it has a bunch of antioxidants which extend its shelf life and increase flavor and taste of other products; thus, making it an ideal ingredient in the various food industries. After roasting, sesame oil becomes resistant to rancidity because of the existence of different natural antioxidants. Moreover, particular roasted flavor of sesame further increases the taste of fried products. Asian countries especially in Africa, sesame seeds are used as seasoning. Likewise, seed oil is used for frying of meat and vegetables and is also used in different confectionery industries. In European countries, sesame oil is used as an alternative to olive oil. Sesame oil is also considered an excellent oil for seasoning salad. It is also used by the Japanese for cooking of various food items (fish). Sesame oil is used as a carrier for drugs and skin softener in the manufacturing of soap and margarine (Begum et al., 2000). Honey from Mexican cactus flower is combined with hulled sesame seed in order to form sesame honey bits. Products like honey puffed kasha seven whole grains and sesame cereal, sesame crackers, sesame seed candy, un-hulled sesame and sesame blues chipsare beingexported in US Health Food and Grocery Stores with sesame seed as an important ingredient. Various food products have sesame seeds as an ingredient e.g. sesame spread, tangerine, cookies, sprouts, bagels, hummus, granola, mustard sauce, broccoli rice, pastry, ginger chicken, green beans and sesame seed sauce. Confectionery items like bread, cakes, rolls, pastries, doughnuts etc. are inexpensive and suitable food items that are prevalent throughout the world. Doughnuts are of different varieties and these are also shaped in the form of balls, stars and fingers. Hot doughnuts may be sprinkled with sugars and spices while cold doughnuts may be filled with chocolate, jam, custard or glazed with icing. Doughnuts raised with yeast contain a light exposed texture. 28 These are considered strong foods having high ratio of consumer acceptability with respect to energy, nutrition and diet. Cereal bars made with peanuts and sesame flour showed strong storage stability because of inherent antioxidants and gave more consumer acceptability as well at storage stability at 37ºC for 15 days (Estevez et al., 2000). Currently, sesame flour and cereal-based bars have been examined against cancer in order to check their therapeutic potential. The commercially available low glycemic index snack bars have proven effective in lowering the risks of prolonged diseases in healthy adults (Miller et al., 2006). In Africa, sesame flour is used in cologne and perfumes. Myristic acid (C14:0) is used as an important element in cosmetics. Sesamin and sesamolin has insecticide and bactericide actions. These are also used as a synergist for pyrethrum insecticides. 2.4. Snacks The term snack was first coined by John Montagu (1718-1792). Snacks are generally regarded as food items meant for consumption between regular meals. Furthermore, these are usually smaller than the regular meals. Currently, “sandwich” is known as world’s most common snack. It is challenging to classify snacks due to their diverse variety, various types of preparatory methods and a variety of ingredients being used to make them (Booth, 1990). Ready-to-eat (RTE) is another common term used these days. Food Standards Agency of UK has defined RTE as any food meant for consumption without more processing or heating (Food Standards Agency, 2011). These are widespread among the consumers over the last decade due to the attractive features like good storage conditions and ease in accessibility of different products (Harper, 1981). These forms of snacks can be consumed as cold or hot. It also explained that there is no need to perform more preparative activities i.e. slicing, portioning, marinating and chopping by the consumer. Now a day, market shelves are filled with energy- dense snacks like breakfast cereals, cereal bars, biscuits, cakes, buns and doughnuts etc. In 18th century in America, snack industry was mainly established on the basis of high consumer demand for cereal and legume based breakfast products. This introduced a lot of new ingredients and invention of hundreds of energy dense snack foods within no time (Fast, 1999). Recently, according to the Euro Monitor Report 2011, the market size in UK has estimated to reach £4.6 billion. The total production was assessed as 448000 tones (Euromonitor, 2011). Unhealthy snacking is one, which provides calories in excessive amount (more than the calorific allowance) like having increased contents of sugar or fat or decreased quantity of 29 essential nutrients i.e. minerals, dietary fiber and vitamins. Unhealthy snacking is generally conditional and it has various forms. The first type is that in which body remains in hunger state for longer time, which further causes increased production of neuropeptides in the brain that lead to the body to be habitual of eating snacks high in sugars. This may lead to obesity and overweight. Second type is the passionate snacking (Cleobury and Tapper, 2014). In this situation, a person eat more than its body requirements due to impact of sentiments like excitement, depression, frustration, stress, love or boredom. This condition also led to obesity and overweight. Third form is consumption for ease. It is a state when people choose calorie- dense snacks in order to prevent binge eating for prolonged period of time. They do not even think that either essential nutrient is present or not. All of these types of snacking are responsible for causing malnourishment (Overby et al., 2012). Another important question is that either the snack is healthy or not? There should be some statistics about the composition of nutrients it contains. DRI (Dietary Reference Intake) was formulated by FDA (Food and Drug Administration of USA) to give data for producers as well as consumers. This is the concentration of nutrients needed every day to overcome deficiency and ability to perform appropriately. Therefore, knowledge about nutrients density is helpful in classifying a healthy or unhealthy snack. Relative nutrient density is the amount of nutrient per unit energy (calorie) of a food and is used as standard in order to compare a snack. Additional term used is the energy density. It is explained as the amount of energy (calorie)/gram of food. A healthy snack is that which provides maximum nutrient and minimum energy density (Raynor et al., 2012). Food selection based on low energy concentration helps to reach satiation at comparatively less calorific value and sustain body weight. 2.4.1. Healthy snacking Customers are very much conscious about their wellbeing and fitness these days. They, therefore, are demanding snacks which can accomplish their desires for eating and provide nutrients, proper health and even more health benefits than that of novel foods (Euromonitor, 2011). Conventional snacks usually have more carbohydrates but they have less quantities of protein, fiber, minerals and vitamins. Moreover, they contain more calories and due to lack of essential nutrients they have been considered as less valuable foods. Now a day, consumer is interested in snacks which are low in sugar and fat, rich in dietary fiber and fortified with minerals and vitamins along with provision of satisfactory hunger. So, the manufacturers must 30 modify the conventional formulations to a new balanced one by the addition of healthy elements (Vitaglione et al., 2008; Altan et al., 2008). This is not minor technical task as the incorporation of these ingredients makes the final product organoleptically objectionable due to increased density, harder making and less expansion. Healthy snacking gives the consumer a stable life through provision of essential nutrients in balanced proportion with the maintenance of the energy value (Fisher et al., 2013). There are five main features which makes a healthy snack i.e. adequacy, balance, calorie control, variety, satiation and satiety. Adequacy means snacks consumed should supply basic nutrients in sufficient amounts as per body necessities. Balance accentuates on nutrients from all groups. Calorie control means that intake of snacks must be responsible for provision of ample energy in such a manner that dietary allowance can be maintained. Diversity means a combination of a variety of constituents which are obtained from various food groups to make snacks further delightful. Satiety and satiation means that there is no need of more as the requirement of the body is fulfilled (Kleef et al., 2012). In this situation, body modify itsmetabolic state by sending signals to the brain that there is no need of more food as the requirement for the food is satisfied. A research was conducted on corn snacks developed through extrusion processing. These were enriched with protein, micronutrients and dietary fiber. Chickpea, defatted soy flour and guar gum were incorporated in corn flour whereas micronutrients like iron, iodine, zinc and vitamin C, A, and folic acid were also added according to their percent daily values. Among the resultant products, fortified snacks containg chickpea and soy (15/100g and 15/100g) were more accepted by the consumers (Shah et al., 2017). Likewise, rice crisps enriched with protein and micronutrient were manufactured by adding 25-40g/100 defatted soy flour and four micronutrients through supercritical fluid extrusion. In the resultant products, protein contents (334-568%) and dietary fiber (571-901%) improved besides 100% retention of added minerals and 50% stability of vitamins (Sharif et al., 2014). Bashir et al. (2017) developed spirulina supplemented rice-soy crisps in which 87-100% minerals, 45-50 vitamin A and 47-52% vitamin C was retained after extrusion processing. These types of products can be used in school nutrition programs especially in developing countries due to better shelf life, palatability, nutrient stability and cost effectiveness. 31 Commonly snacks are interrelated with children’s diet. In the previous era, snacks were prominently explored by various countries and used by governmental and non-governmental societies for elevating the nutritional requirements of school going children. Likewise, manufacturing of iron-based snacks was encouraged to enhance the iron status in the females. A new evolving notion is the “snack in terms of food for geriatrics”. Researches have shown that if the caloric requirements of 25-70 years people are calculated, there is decrease of 600- 800 Kcal/day for women and 1,000-1,200 Kcal/day for men. The main mortality causing factors for the elderly is the inadequate intake of carbohydrates, protein, vitamins, minerals and fats. Resultantly, there is an undesirable loss in body weight as well as micronutrient deficiencies which further elevate risks of chronic diseases. With the help of special formulations, the nutritional needs of these groups can be fulfilled (Zizza et al., 2007). 2.5. Conclusion In this era of knowledge and better means of communication, the basic reasons for the manifestation of malnutrition are lack of resources, ineffective policies & programs and poor compliance of commitments. We have now better expertise to produce nutritious products as advance strategies such as fortification, supplementation and enrichment have made it very easy to develop nutrient-dense products. However, we are confronted with issues of poor & ineffective coordination, communication, supply and demand. Private and public sectors required to interact and equally reinforce their struggles to minimize the degree of this global issue at national level. Globally, this is the right time to initiate and eliminate malnutrition by involving all stakeholders from academia, research, farming, industry, health and policy sectors. For this purpose, communication and resources from public sector as well as new technologies through intended programs could be vital. Food-based malnutrition tackling tactics should be given due weightage. Nutrient-dense conventional crops should made part of regular diets. In this way people could attain their full public right to live a healthy life. In return they can work more powerfully for the social and economic welfare of mankind. 32 CHAPTER 3 MATERIALS AND METHODS Research work for the development of protein enriched nutrient-dense snack was performed in the Food and Nutrition Laboratory at the National Institute of Food Science and Technology (NIFSAT), University of Agriculture, Faisalabad, Pakistan. Sesame fortified doughnuts were developed and analyzed for physicochemical analysis, nutritional composition, storage stability and consumer acceptance. Furthermore, animal trial was conducted using Sprague Dawley rats for the biological evaluation of developed products. Subsequently, doughnuts were served to school going children to determine the impact of nutrient enrichment on growth and development through anthropometric methods and blood analysis. The materials and protocols followed in the study are discussed below: 3.1. Procurement and Preparation of Raw Materials Seeds of Pakistani white (TH-6, TS-3, TS-5 and Til-89) and black cultivars (S-122, S-117, S- 131 and Latifi) were procured from Oil Seed Section, Ayub Agricultural Research Institute (AARI), Faisalabad and Agriculture Research Institute (ARI), Tandojam, Pakistan. Micronutrients pre-mix comprising of folic acid, Zn, Fe, vitamin A and C and was procured from Fortitech Inc. Schenectady, NY, USA. The HPLC and analytical grade reagents, standards and diagnostic kits were obtained from the Merck (Merck KGaA, Darmstadt, Germany), Sigma-Aldrich (Sigma-Aldrich, Tokyo, Japan) and Bioassay (Bioassays Chemical Co., Rodgau, Germany). Rats (Male Sprague Dawley) were obtained from the National Institute of Food Science and Technology (NIFSAT), Faculty of Food, Nutrition and Home Sciences (FFNHS), University of Agriculture, Faisalabad (UAF). Manual cleaning of sesame seeds was done to remove the physical impurities followed by oil extraction through Manual Oil Press (Carver Inc., Wabash, IN, USA). Subsequently, sesame cake samples were ground to get sesame cake flour. For further analysis and utilization, all these samples were packed in polyethylene zip bags and stored at 25ºC. 33 3.2. Analysis of Raw Materials All sesame flour samples were analyzed for proximate composition, minerals, amino acids, antioxidant potential and functional components using their respective methods. The brief explanation of each is as under: 3.2.1. Proximate composition Defatted sesame flour samples were analyzed for moisture (AACC Method No. 46-30), crude protein (Method No. 46-10), crude fat (Method No. 30-25), crude fiber (Method No. 32-10), ash (Method No. 08-01) and nitrogen free extract (NFE) according to their respective methods as described in AACC (2000). 3.2.1.1. Moisture Hot air oven (Lab Tech. LDO-150N, Daihan Labtech Co. Ltd., Kyonggi-DO, Korea) was used to determine the moisture contents. In China dish, 5g sample was taken and placed at 105±5°C to achieve the constant weight. The formula used for calculation was: Wt. of original sample (g) – Wt. of dried sample (g) Moisture (%) = ------× 100 Wt. of original sample (g) 3.2.1.2. Crude protein Kjeltech apparatus (Technick GmbH, Behr Labor, Germany) was used to determine the nitrogen contents. For this purpose, sample (2g) was digested with 25mL H2SO4 (conc.) and digestion mixture (5g) for 4-6hrs till a light green color was obtained after filtration and dilution (volume was made upto 250mL), distillation (10mL) was performed using 4% boric acid (H3BO3) and 40% sodium hydroxide (NaOH). Finally, titration was done with 0.1N sulphuric acid (H2SO4) till light pink endpoint to get % nitrogen. Vol. of 0.1N H2SO4 used (mL) × Vol. of dilution (mL) × 0.00l4 Nitrogen (%) = ------× 100 Wt. of sample (g) × Vol. of diluted solution used (mL) Crude protein (%) = Nitrogen % × 6.25* *= Conversion factor 6.25 for sesame 34 3.2.1.3. Crude fat Fat content was determined through Soxlet apparatus (98-1-B, PCSIR, Pakistan). In extraction thimble, 2g sample (oven dried) was placed and attached to the Soxlet assembly. Solvent (250mL of n-hexane) was used for continuous refluxing of the sample. The adjustment of the solvent for the extraction of fat was done at the rate of three to four drops per second upto 2-3 hrs. Afterwards, thimble was detached with subsequent drying of sample in a hot air oven at 105±5°C for 1h and weighed. Loss in weight Crude fat (%) = ------x 100 Wt. of sample (g) 3.2.1.4. Crude fiber Fibertech (Labcono Corporation, Kanas, USA) was used to determine the fiber content in moisture free sample. The sample (2g) was digested in 1.25% boiling H2SO4 for 30 minutes. The sample was made acid free by draining out the acid as well as washing with distilled water (three times). Subsequently, samples were digested with 1.25% boiling NaOH for half hour. The sample was made alkali free by following the same procedure of filtration and washing. In pre-weighed crucible, the residue was dried at 105oC±5oC in a hot air oven. Finally, placement of charred samples was done in a muffle furnace at 550±5°C, to obtain the grayish white color. The loss in weight was calculated by weighing the ash obtained after burning. The formula used to calculate the fiber content is as under: Weight of oven dried sample (g) – Weight of Ash Crude fiber (%) = ------x 100 Wt. of sample (g) 3.2.1.5. Ash content 3g oven dried sample was burnt on flame before cremating in Muffle Furnace at 550°C (MF- 1/02, PCSIR, Pakistan) to obtain the residue having grayish white color. Subsequently, sample was cooled in desiccator, weighed and % ash was determined: Weight of ash in sample (g) Ash (%) = ------x 100 Weight of sample (g) 35 3.2.1.6. Nitrogen free extract Nitrogen free extract (NFE) was calculated according to the following expression: NFE = 100 - (%Moisture+ %Crude protein+ %Crude fat+ %Crude fiber+ %Ash) 3.2.2. Mineral contents Sesame cake flour samples were probed for mineral profile after wet digestion (AOAC Method No. 975.03B). Na and K contents were analyzed using Flame Photometer-410 (Sherwood Scientific Ltd., Cambridge, UK) while Mg, Ca, P, Fe and Zn contents were determined using Atomic Absorption Spectrophotometer (Varian AA240, Varian Medical Systems, Australasia Ltd., Belrose, Australia). First digestion of sample (0.5g dried) was done at 60-70°C for 20min on hot plate using HNO3 (10mL) in a 100mL conical flask followed by second digestion with HClO4 (5mL) at 190°C till the contents in the flask become transparent. Digested sample was diluted in 100mL volumetric flask using deionized water and then filtration was performed. Samples of known strengths were run to measure the standard curve (AOAC, 2016). 3.2.3. Amino acid profile Ion-exchange chromatography with automatic Amino Acid Analyzer, (Hitachi L8500, Tokyo, Japan) was used to determine amino acids in sesame flours following the method described by Walsh and Brown (2000). The defatted ground sample (30mg) was taken along with 5μmol norleucine and 5mL of 6M HCl into glass ampoules. These were further emptied by using liquid N2, vacuum-packed and dried in an oven (110°C) for one day. After cooling and filtration, samples were dried under vacuum in rotary evaporator (40oC). For neutral and acidic amino acids, 5μL of dilution was made with acetate buffer having pH 2.2 whereas for basic amino acids, 10μL dilution was made. Subsequently these dilutions were spread over the cassette of amino acid analyzer. The complete sequence of amino acids was quantified by comparing the peak of each amino acid with its standard. 3.2.4. Antioxidant potential 3.2.4.1. Total phenolic contents Total phenolics of sesame flour samples were analyzed through UV/Vis spectrophotometer (VIS-1100Spectrophotometer, Biotechnology Medical Services, Medifield, MA, USA). The extract (125μL) was combined with Folin-Ciocalteau (FC) reagent (125μL) by adding distilled water (500μL) and endorsed to stand for 5min at 22°C. 4.5mL solution of sodium bicarbonate (7%) was poured in the mixture after following the resting duration. The absorbance of each 36 sample was observed at 765nm after 90min against the control. The unit used to express the total polyphenols was Gallic acid equivalent (mg gallic acid/g) (Pourmorad et al., 2006). 3.2.4.2. DPPH free radical scavenging ability Free radical scavenging activity assay was analyzed by following the method of Muller et al. (2011). For the purpose, sample solution (1mL), 0.5mL of 0.3M Methanolic 1,1-diphenyl- picrylhydrazyl (DPPH) solution and standard or blank (ethanol/n-hexane 1 + 1, v/v) were shaken at 25±1 °C, 1000rpm in a thermo shaker. The absorbance was taken at 540nm after 15min and inhibition was measured by the given formula: Reduction of absorbance (%) = [(AB - AA) / AB] × 100 Where; AB = absorbance of blank sample (t = 0 min) AA = absorbance of tested extract solution (t = 15 min) 3.2.4.3. Antioxidant activity (β-carotene bleaching method) Coupled oxidation oflinoleic acid and beta-carotene was assessed for total antioxidant activity of the sesame flour samples (Kenariet al., 2013). 2mg of -carotene was dissolved in chloroform (20mL) and Tween-20 (400mg). After chloroform removal, emulsion (3mL) was added in prepared sample (0.10mL). It was further placed in a water bath (120min). The oxidation of β-carotene was determined at absorbance of 470nm. 3.2.5. Functional components Samples were extracted according to the modified methods of Rangkadilok et al. (2010) and Amber et al. (2012). Ground sample (2g) was passed through a screen (0.25mm) and 200mg weighed flour was shifted into a cuvette (10mL) having 80% ethanol (5mL). Centrifugation of samples (vortex-mixed) was done at 17500g for 5min. In volumetric flask, the supernatant was transferred, and the residue was extracted again with 80% ethanol (5mL). Filtration of all extracted solutions was done through filter of nylon membrane (0.45μm pore size) before performing HPLC analysis. Sesame cake flours functional components like sesamin, sesamolin and sesamol were examined according to procedures described by Shahidi et al. (2006) and Kaya et al. (2012) by using high-performance liquid chromatography (Perkin Elmer Series 200 HPLC Systems, Perkin Elmer Life and Analytical Sciences, Shelton, USA).A reversed-phase C18 column (5μm particle sizes, 150 × 4mm) was used. Quantification and evaluation was performed on Total Chrome Software. The mobile phase i.e. methanol/deionized water 37 (80:20%) was passed at a rate of 0.8mL/minute with constant temperature of 40°C. The injection volume was 10μl; elution rate was 2.0mL/min. For the determination of compounds in the samples, the working standard solutions were analyzed at 290nm with the samples. Peak areas and retention time of both samples as well as standards were used to quantify the respective component. 3.3. Preparation of Sesame Cake Flour Supplemented Doughnuts Sesame cake flours were supplemented in wheat flour in different proportions (Table 3.1) to prepare sesame cake flour supplemented doughnuts by following modified methods of Oke et al. (2018) and Nsabimana et al. (2018). Micronutrient premix (vitamin A, B9, Fe, Zn, vitamin A and C) was added according to % daily values in all formulations during the blending process (Annexure-I). All ingredients were mixed and kneaded to form dough followed by sheeting and molding (round shaped doughnuts of specific size with a hole inside it. Furthermore, frying was done at 175°C and finally all samples were packed in polyethylene zip bags for further analysis. 3.4. Sensory Evaluation of Sesame Cake Flour Supplemented Doughnuts Sensory analysis of the doughnuts w as performed using nine-point hedonic score system (1=disliked extremely up to 9=liked extremely) for various attributes like color, taste, aroma, chewability, texture, mouthfeel and overall acceptability (Annexure-II) by a sensory panel using the guidelines of Meilgaard et al. (2007). The judges were offered doughnuts in polystyrene plates (white) at 25°C. Three-digit codes were used to label them and offered in distinct booths provided with white florescent light. Successively, the evaluators were requested to score the snacks. Before each assessment, the judges were served with plain crackers and water to clean mouth and neutralize their taste buds. Furthermore, panelists’ names were not revealed in order to retain privacy. 38 Table 3.1. Formulations used in study (g/100g) Treatments *Sesame cake flour (%) T0 - T1 10 T2 20 T3 30 T4 40 T5 50 T0 = Doughnuts without supplementation act as control T1 = Doughnuts supplemented with 10% of white sesame flour of S-122 T2 = Doughnuts supplemented with 20% of white sesame flour of S-122 T3 = Doughnuts supplemented with 30% of white sesame flourof S-122 T4 = Doughnuts supplemented with 40% of white sesame flour of S-122 T5 = Doughnuts supplemented with 50% of white sesame flour of S-122 *Same treatment plan was repeated for all remaining white and black sesame cultivars 39 3.5. Selection of Best Sesame Cultivar Based on consumer acceptability, two best formulations along with control were selected for assessment of nutrient composition, storage stability, biological evaluation and efficacy studies. 3.6. Analysis of Sesame Cake Flour Supplemented Doughnuts All selected doughnut formulations were studied for proximate composition, minerals, water activity, texture, color and calorific value using their respective procedures. 3.6.1. Proximate composition Sesame cake flour supplemented doughnuts were examined for proximate composition following methods as described earlier (AACC, 2000). 3.6.2. Mineral Contents All samples were studied for Na, K, Ca, Mg, P, Fe and Zn after wet digestion using Flame Photometer-410 and Atomic Absorption Spectrophotometer by following methods of AOAC (2016). 3.6.3. Water activity An electronic Hygropalm water activity meter (Hygropalm AW1, Rotronic AG, Bassersdorf, Bulach, Switzerland) was used to estimate water activity of the samples (AOAC, 2016). 3.6.4. Texture analysis The doughnut samples were evaluated for textural profile by using a texture analyzer (TA-XT Plus, Stable Micro Systems Ltd., Surrey, UK) equipped with Texture Expert version 1.22 Software. The instrument was having a load cell (5kg) and a cylinder-shaped probe (20mm). The firmness test was performed at 1.0mm/s and complete information of the sample without excessive densification was provided by 50% strain level. Texture characteristics such as hardness, cohesiveness, springiness and chewiness were determined by using double compression deformation curve of the samples as described by Mazumder et al. (2007). 3.6.5. Color measurement Color was determined as described by Sharif et al. (2014) with some modification using color meter (Chroma Meter CR-400, Konica Minolta, Sensing Inc. Japan). The L*-value in color system showed luminance/brightness with 0-100 (darkness-brightness); a*-value depicts the 40 green and red colors in the range from -100 to 0 and 0 to -100; b*-value measures blue (-100- 0) and yellow (0-100). 3.6.6. Calorific value Calorific value of the doughnuts was evaluated by using oxygen bomb calorimeter (C2000 Basic, IKA-WERKE GmbH & CO., Germany) as reported by Krishna and Ranjhan (1981). In decomposition vial, sample (0.5g) was placed with the support of a cotton thread and ignition wire. Afterwards, decomposition vial was tightened with the screw cap and directed into the filler head. The measuring cell cover was closed by pushing the start button. The sample was burnt through electric spark within the vial. The produced heat was noted by the C5040 CalWin software (IKA-Werke, Germany) in a graphical form indicating the temperature versus time which reflects the number of calories/gram in a test sample. 3.7. Storage Study All selected formulations were stored for 7 days at refrigerated temperature (4°C) and examined for peroxide value (AOAC, 2016), thiobarbituric acid number (Kirk and Sawyer, 1991) and microbiological analysis (AACC, 2000) at the initiation, middle and end of the storage. 3.7.1. Peroxide value The peroxide value of samples was calculated in term of iodine formed by the reaction of iodide ions and hydrogen peroxide by following AOAC Method No. Cd 8-53 (AOAC, 2016). Sample (5g) was taken in a flask (250mL) followed by addition of glacial acetic acid- chloroform (3:2 v/v) solution. The flask was spun for a while in order to completely dissolve the oil in the solvent mixture. 0.5mL of freshly prepared saturated solution of potassium iodide (KI) was added in the flask and the contents were titrated against standard solution (0.1N sodium thiosulphate i.e. Na2S2O3) with constant shaking to eliminate the yellow color. Then starch solution (0.5mL) was added as an indicator and titration was continued with vigorous shaking till the fading of blue color. The reading for blank was separately observed. It was calculated by using the following formula: (B – A) x N x 1000 Peroxide value (mEq/Kg of fat) = ------x 100 Weight of oil sample (g) 41 B = Volume of Na2S2O3 used for blank A = Volume of Na2S2O3 used for sample N = Normality of Na2S2O3 3.7.2. Thiobarbituric acid number (TBA No.) In distillation flask, sample (10g) was taken and distillate (5mL) was obtained after heat treatment in test tube (covered with glass stopper) alongwith TBA reagent (0.2882g/100 of 90% glacial acetic acid) and heated in water bath for 35 minutes along with a blank sample. After cooling, absorbance (D) was observed at wavelength of 538nm.TBA no. was estimated by using the following relationship: TBA no. (mg malenaldehyde/Kg sample) = 7.8 x D 3.7.3. Mold growth It was monitored by following AACC Method No. 42-50 (AACC, 2000). Media (potato dextrose agar) was sterilized in autoclave. Sterile spoon was used to take the sample (1g) and shifted in sterile blender along with sterile (9mL) buffered phosphate diluent and mixed thoroughly at low speed for 1-2 min. 1.0mL of each dilution was shifted into labelled duplicate Petri dishes. Subsequently, 15.0mL of media was poured in the plates immediately. It was mixed well and endorsed to solidify. For series of samples, dilutions of sample were mixed with the rotation of plates on the surface. Before inverting plates, these were solidified and incubated at room temperature. Colonies were counted (<50 colonies) on the plates by multiplying with dilution factor in the end. The unit used was total plate count/gram. 3.8. Biological Evaluation Biological quality of doughnuts was estimated by feeding diets comprised of selected formulations to male Sprague Dawley rats along with control (soy protein). Isonitrogenous diets (having the final protein content upto 10%) were made (Mensa-Wilmot et al., 2001). Nitrogen-free mixture used in study was comprised of sucrose, cellulose and corn starch. 3.8.1. Housing of rats Male Sprague Dawley rats (n=15). The rats were distributed into 3 groups (5 in each) and served with their basic routine diet for seven days. Subsequently, groups were served with respective diets for 10 days (Table 3.2). 42 Table: 3.2. Experimental diets Diet Constituents Diet-1 Diet-2 Soy diet Sesame based doughnuts (S1) 87.37 0.0 0.0 Sesame based doughnuts (S2) 0.0 56.80 0.0 Soy protein 0.0 0.0 10.69 Corn oil 5.0 5.0 5.0 Mineral mixture 5.0 5.0 5.0 Vitamin mixture 1.0 1.0 1.0 N-free mixture 1.63 32.2 79.31 Total diet weight 100 100 100 Values are shown as gram; all the diets contain ~10% protein 43 During the experimental period, the conditions were made constantat 23±2°C temp. and 50±5% humidity with 12-hr light-dark cycle. Water intake, feed intake, body weight gain, spilled diet and feces were collected daily during the trial. Finally, rats that were kept fasted overnight, killed and their body remains were incinerated in an electric oven. 3.8.2. Protein quality evaluation Growth study parameters i.e. net protein ratio (NPR), protein efficiency ratio (PER) and relative net protein ratio (RNPR) were calculated through the results of body weight gain and gross feed intake. Whereas, nitrogen balance study parameters i.e. biological value (BV),true digestibility (TD) and net protein utilization (NPU) were studied through the calculation of nitrogen content from the analysis of spilled diet, feces, urinary outputs and dried rat bodies as described by (Pellet and Young 1980; Ingbian and Adegoke 2007). 3.9. Efficacy Trial After establishing nutrient profile, storage stability and biological evaluation, selected compositions of doughnuts were served to school aged children (n=45) for 60 days (study interval) during the school days. The study was carried out in Govt. Primary School, Chak # 529/EB, which is located in low socio-income area of district Vehari, Pakistan. Institutional Biosafety/Bioethics Committee of the University of Agriculture, Faisalabad gave permission to conduct the study (Annexure-III). 3.9.1. Acceptability Study Short term feeding trial was done to analyze the children acceptability for doughnuts as stated by Thathola and Srivastava (2002). Selected children of all groups were served with doughnuts along with a feedback form to observe the response of acceptability. Questionnaire was framed on the basis of five-point hedonic scale (1 = rejected; 5 = highly accepted). For further study, children with great acceptance were selected. 3.9.2. Ethical considerations Before the commencement of trial, short lectures and presentations were given to the school children, their teachers as well as parents in native language i.e. Urdu and Punjabi to provide knowledge about the product and importance of the entire study. During the trial period, parents and teachers were also asked to keep noticing any unusual observation related to general health. Subsequently, inclusion of respondents in the study was made sure by their 44 physical examination through a physician to check either they are strong enough to accomplish this task or not. The factors for the exclusion criteria were, severe anemia, physical disability, failure of body organ, frequent infections, food allergies, intake of nutritional supplements, participation of child in any study related to nutrition in the past and chances of leaving the study by the child’s family. In Pakistan, about 35-40% Helminthes infection is prevalent, so 400mg of albendazole (Crystolite Pharmaceuticals) were given to every child to deworm them before initiating the trial (Kumar and Rajagopalan, 2006). Finally, written Informed Consent Form (Annexure-IV) was taken from parents as well as school authorities assuring the presence of each respondent in the intervention after complete satisfaction on the study. 3.9.3. Ethical trial School children (n=45) were arbitrarily distributed into 3 groups (15 in each), served with 100g doughnuts during mid-day break on each school day (Annexure-V). Experimental groups were served with fortified doughnuts having 10 and 20% sesame flours, respectively. While, the control group was served with fortified doughnuts only. Full biochemical and nutritional assessment along with micronutrient status were observed at baseline, middle and at the end of the study. Study decorum was made reliable among the respondents. 3.9.4. Anthropometric examination Anthropometric parameters were measured among all the children during the study. Height and weight scale were used to measure them individually. Furthermore, body mass index was also evaluated. Similarly, mid upper arm circumference of the children was measured by using special three colored (Yellow, Green and Red) tapes after removal of additional clothing, taking child’s left arm and all measurements were taken close to 0.10cm and immediately noted (Skelton and Rudolph 2007; Aziz et al. 2012). Occipital-frontal head circumference was measured by using non-elastic measuring tape (Lee and Nieman, 2002). 3.9.5. Blood sampling Blood samples of the children were taken by following the method of World Health Organization. It was instantly shifted to vials and conveyed to the laboratory in an ice box for further tests (WHO, 2010). 3.9.6. Hematological analysis 45 Complete blood count was done to determine red blood cells, white blood cells, haematocrits, hemoglobin, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelet count, lymphocytes, neutrophils, eosinophils and monocytes. Analysis of serum zinc, ferritin, blood sodium, chloride and potassium were performed for the nutritional assessment. Renal and liver function tests of the human subjects were performed to estimate the possible adverse health effects of the test diets. 3.10. Statistical Analysis The collected data obtained from all parameters were statistically analyzed using Statistical Package (SPSS). Level of significance was evaluated by analysis of variance techniques (ANOVA). Means were compared through Tukey’s honest significance test (Steel et al., 1997). 46 CHAPTER 4 RESULTS AND DISCUSSION The current study was performed to develop protein enriched and nutrient-dense doughnuts by food supplementation and fortification methodologies. Sesame was used to increase the protein contents. Sesame varieties (TH-6, TS-5, TS-3, Til-89, S-122, S-117, S-131, Latifi) were characterized by assessing proximate composition, minerals, amino acids, antioxidant potential and functional components. Afterwards, protein enriched and micronutrient fortified doughnuts were developed and analyzed for chemical composition, minerals, water activity, texture, color, calorific value, consumer acceptability and storage study parameters like peroxide value, thiobarbituric acid number and microbiological analysis. Based on consumer acceptability, best selected formulations were further used for protein quality evaluation through Sprague Dawly rats. Furthermore, impact of fortification on serum profile and nutrient availability was evaluated through feeding trial in school aged children. The results of above mentioned parameters are described underneath: 4.1. Analysis of Sesame Cake Flours Sesame flours of different cultivars were analyzed for proximate composition, minerals, amino acids, antioxidants, and functional components using their respective methods. The results of each parameter are as under: 4.1.1. Proximate Composition Mean squares for proximate analysis of sesame flours of white and black cultivars exhibited significant differences with respect to moisture, crude protein, crude fat, crude fiber, ash, and nitrogen free extract (Table 4.1). Means for moisture contents of different sesame cultivars (Table 4.2) were ranged from 7.23±0.32 to 11.82±0.38%. Among the different cultivars, the highest moisture (11.82±0.38%) was observed in Latifi followed by S-131 (11.32±0.14%) and S-117 (10.87±0.34%). The lowest moisture (7.23±0.32%) was in TH-6 followed by TS-5 (8.19±0.17%). Comparatively, the moisture content was low in white cultivars than that of black ones. Means for crude protein content (Table 4.2) revealed values ranged from 33.59±0.08 to 38.97±0.28%. The highest protein (38.97±0.28%) was observed in TH-6 followed by TS-5 (38.21±0.40%), TS-3 (37.15±0.50%) and (36.04±0.13%) in Til-89. 47 Table 4.1. Mean squares for proximate composition of sesame flours of different cultivars SOV Df Moisture Crude protein Crude fat Crude fiber Ash NFE Varieties 7 7.7936** 11.186** 23.205** 11.105** 11.498** 30.983** Error 16 0.1965 0.357 0.325 0.086 0.127 1.539 Total 23 ** = Highly significant (P<0.01); Df = Degree of freedom 49 Table 4.2. Proximate composition (%) of sesame flours of different cultivars Varieties Moisture Crude Protein Crude Fat Crude Fiber Ash NFE TH-6 7.23±0.32e 38.97±0.28a 12.83±0.33b 5.84±0.16d 9.93±0.18a 25.30±0.36cde TS-5 8.19±0.17de 38.21±0.40ab 11.90±0.26bc 5.09±0.11d 9.15±0.22ab 27.79±0.51bc TS-3 8.72±0.25d 37.15±0.50bc 11.15±0.38cd 4.23±0.12e 8.65±0.28bc 29.94±0.71ab Til-89 9.32±0.19cd 36.04±0.13cd 10.25±0.52d 3.62±0.05e 7.66±0.18c 32.42±1.45a S-122 10.16±0.14bc 35.33±0.38de 17.23±0.29a 8.77±0.14a 6.33±0.18d 22.75±0.52e S-117 10.87±0.34ab 34.74±0.51def 16.75±0.39a 8.18±0.23ab 5.70±0.27de 23.79±0.18de S-131 11.32±0.14ab 34.21±0.18ef 16.20±0.18a 7.80±0.20bc 5.16±0.17e 25.33±0.56cde Latifi 11.82±0.38a 33.59±0.08f 15.83±0.11a 7.25±0.25c 4.73±0.11e 26.67±0.69bcd Means having same letters are statistically non-significant (P>0.05) Means ± S.D 50 The lowest protein content (33.59±0.08%) was found in Latifi followed by S-131 (34.21±0.18%). Statistical results indicated that protein contents varied significantly among different sesame cultivars. White varieties contain more protein as compared to black counterparts. Means for crude fat content (Table 4.2) showed values ranged from 10.25±0.52 to 17.23±0.29%. Among the white and black cultivars, the highest fat content (17.23±0.29%) was observed in S-122 followed by S-117 (16.75±0.39%), whereas the lowest fat (10.25±0.52%) was found in Til-89 followed by TS-3 (11.15±0.38%). Results revealed that fat contents of white sesame were significantly lower than black sesame. Means for crude fiber content (Table 4.2) showed values ranged from 3.62±0.05 to 8.77±0.14%. Among the different cultivars, the highest fiber content (8.77±0.14%) was observed in S-122 followed by S-117 (8.18±0.23%) and S-131 (7.80±0.20%). Likewise, the lowest fiber content (3.62±0.05%) was in Til-89 followed by TS-3 (4.23±0.12%). It is obvious from the results that white sesame contains less fiber contents than black ones. Means for ash content (Table 4.2) revealed values ranged from 4.73±0.11 to 9.93±0.18%. The highest ash content (9.93±0.18%) was noted in TH-6 followed by TS-5 (9.15±0.22%), and TS-3 (8.65±0.28%), respectively. The lowest ash content (4.73±0.11%) was noticed in Latifi. Overall, ash content in white sesame flour was significantly lower than black sesame cultivars. Means for NFE content (Table 4.2) showed values ranged from 22.75±0.52 to 32.42±1.45%. Regarding white and black varieties, the highest NFE content (32.42±1.45%) was observed in Til-89 followed by TS-3 (29.94±0.71%), whereas the lowest NFE (22.75±0.52%) was found in S-122 followed by S-117 (23.79±0.18%). The variation in the proximate composition of sesame flours of white and black cultivars were might be due to genetic variations, different climatic conditions, milling actions, and agronomic practices during the cropping season. Sesame seed contains high quality protein due to presence of all essential amino acids in balanced proportions. Variations in protein contents among the selected varieties were might be due to several factors including type of cultivar, fertilization level, environmental aspects (alkalinity and salinity, diseases attack, temperature,) location of growing areas, conditions and seasonality. In a study, sesame flour supplemented high protein and energy food bars were developed. The results showed that defatted sesame flour of white sesame cultivar (TH-6) contains 2.19% moisture, 51.5% crude protein, 1.49% crude fat, 3.46% crude fiber, 6.15% ash and 45.56% nitrogen free extract 51 (Abbas et al., 2016). According to USDA National Nutrient Database for Standard Reference Library, 100 g edible portion of dry decorticated andpartially defatted sesame flour contains water (6.61 g), protein (40.32 g), fat (11.89 g), carbohydrate (35.14 g) and zero cholesterol (USDA, 2018). In another study, sesame protein concentrates were developed from sesame meals. The proximate composition of sesame meal revealed varying amounts of moisture content (6.8%), crude protein (36.24%), crude fiber (3.46%), ash (6.15%), crude fat (9.5%) and NFE (37.85%), respectively (Onsaard et al., 2010). Earlier findings of a study conducted by Egbekun and Ehieze (1997) have indicated higher protein contents in defatted sesame flours that can be supplemented in low protein flours of cereals for the preparation of infant foods and further used for growth and development of children. Furthermore, as a result of removal of crude fat from 48.8 to 10.9%, fiber content was increased from 3.6 to 5.8% alongwith increase in the contents of ash from 5 to 6.93%, respectively. In another research, defatted sesame meal was prepared by (Jimoh et al., 2011) for African catfishas a replacer for soybean meal. The proximate composition of defatted sesame meal exhibited presence of moisture (10.58%), crude protein (38.32%), fat (12.70%), fiber (5.09%), ash (4.28%) and carbohydrates (30.01%). 4.1.2. Mineral contents Mean squares for mineral contents of sesame flours of white and black cultivars (Table 4.3) showed significant differences with respect to sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe) and phosphorus (P) contents. Means for sodium (Na) content (Table 4.4) showed values ranged from 29.24±0.82 to 60.12±0.89mg/100g. Among the different cultivars, the highest sodium content was observed in TH-6 (60.12±0.89mg/100g) followed by TS-5 (58.77±1.07mg/100g), TS-3 (56.32±0.73mg/100g) and Til-89 (54.12±1.21mg/100g). The lowest sodium content (29.24±0.82mg/100g) was noted in Latifi followed by S-131 (33.61±0.97mg/100g). Means for potassium (K) content (Table 4.4) showed values ranged from 52.78±1.20 to 81.79±1.62mg/100g. Among the different cultivars, S-122 showed the highest potassium content (81.79±1.62mg/100g) followed by S-117 (78.23±1.86mg/100g), S-131 (73.58±1.00mg/100g) and (70.45±1.89mg/100g) in Latifi. The lowest potassium content (52.78±1.20mg/100g)was observed in Til-89 followed by TS-3 (57.43±1.61mg/100g). 52 Table 4.3. Mean squares for mineral contents of sesame flours of different cultivars SOV Df Sodium Potassium Calcium Magnesium Zinc Iron Phosphorus Varieties 7 443.61** 307.04** 435.74** 261.81** 48.905** 19.381** 433.99** Error 16 3.770 7.080 7.320 5.350 0.345 0.069 3.050 Total 23 ** = Highly significant (P<0.01); Df = Degree of freedom 53 Table 4.4. Mineral contents (mg/100g) of sesame flours of different cultivars Varieties Sodium Potassium Calcium Magnesium Zinc Iron Phosphorus TH-6 60.12±0.89a 67.32±1.01cd 62.48±2.43c 59.23±1.48a 13.47±0.35c 7.23±0.14c 63.47±1.11a TS-5 58.77±1.07ab 61.62±1.78de 56.67±1.24cd 56.87±1.95ab 11.87±0.21c 6.58±0.14cd 57.58±1.22b TS-3 56.32±0.73ab 57.43±1.61ef 52.56±0.89de 53.29±0.77ab 9.25±0.32d 4.34±0.12e 53.24±1.51bc Til-89 54.12±1.21b 52.78±1.20f 46.82±0.55e 50.46±1.99bc 7.23±0.16e 2.21±0.06f 51.69±1.05c S-122 42.56±1.89c 81.79±1.62a 80.21±1.90a 44.42±0.92cd 19.53±0.36a 10.21±0.30a 42.82±0.99d S-117 38.43±0.96cd 78.23±1.86ab 76.21±1.39ab 41.83±1.17de 17.06±0.61b 8.36±0.10b 38.21±0.84de S-131 33.61±0.97de 73.58±1.00bc 73.32±1.28ab 37.23±0.77ef 15.46±0.11b 6.02±0.13d 33.68±0.31ef Latifi 29.24±0.82e 70.45±1.89c 70.45±1.94b 33.45±0.95f 12.21±0.32c 4.23±0.10e 29.67±0.49f Means having same letters are statistically non-significant (P>0.05) Means ± S.D 54 Means for calcium (Ca) content (Table 4.4) revealed values ranged from 46.82±0.55 to 80.21±1.90mg/100g. Among the different cultivars, S-122 (80.21±1.90mg/100g) showed the highest calcium content followed by S-117 (76.21±1.39mg/100g) and S-131 (73.32±1.28mg/100g). The lowest calcium content (46.82±0.55mg/100g) was found in Til-89 followed by TS-3 (52.56±0.89mg/100g). Means for magnesium (Mg) content (Table 4.4) showed values ranged from 33.45±0.95 to 59.23±1.48 mg/100g. Among the different cultivars, the highest magnesium content was observed in TH-6 (59.23±1.48mg/100g) followed by TS- 5 (56.87±1.95mg/100g), TS-3 (53.29±0.77mg/100g) and Til-89 (50.46±1.99mg/100g). The lowest magnesium content was found in Latifi (33.45±0.95mg/100g) followed by S-131 (37.23±0.77mg/100g). Means for zinc (Zn) content (Table 4.4) showed values ranged from 7.23±0.16 to 19.53±0.36mg/100g. Among the different cultivars, the highest zinc content (19.53±0.36mg/100g) was found in S-122 followed by S-117 (17.06±0.61mg/100g). The lowest zinc content (7.23±0.16mg/100g) was observed in Til-89 followed by TS-3 (9.25±0.32mg/100g). Means for iron contents showed values ranged from 2.21±0.06 to 10.21±0.30mg/100g (Table 4.4). Among the different cultivars, S-122 showed the highest value (10.21±0.30mg/100g) of iron followed by S-117 (8.36±0.10mg/100g) and S-131 (6.02±0.13mg/100g) whereas the lowest iron (2.21±0.06mg/100g) content was observed in Til-89 followed by TS-3 (4.34±0.12mg/100g). Means for phosphorus (P) contents (Table 4.4) exhibited the highest phosphorus content (63.47±1.11mg/100g) in TH-6 followed by TS-5 (57.58±1.22mg/100g), and TS-3 (53.24±1.51mg/100g). The lowest phosphorus content (29.67±0.49mg/100g) was found in Latifi followed by S-131 (33.68±0.31mg/100g). The variation in the mineral contents in sesame flours of white and black cultivars were might be due to slight differences in degree of milling, the type of soil where the plants were gown, different agro-climatic conditions and genetic makeup. In addition, the method applied, and instruments used for screening of minerals might also resulted in small differences. The results are in conformance with the study conducted on the raw, defatted and protein concentrate of sesame flour. The results of this study exhibited, greater degree of differences among the samples for potassium (72.61mg/100g), Na (59.88 mg/100g), Mg (55.68 mg/100g), Ca (63.42 mg/100g), Zn (17.29 mg/100g), Mn (6.81 mg/100g), Fe (7.26 mg/100g) and Cu (37.56 mg/100g). These values of essential macro and micro-minerals obtained from the defatted 55 sesame flours can satisfy the consumer needs (Ogungbenle and Onoge, 2014). Findings of another research conducted in Ethiopia on the mineral and anti-nutritional contents of different varieties of sesame seed revealed the highest phosphorus contents (660.61-867.0 2mg/100g) followed by potassium (610.15-808.65mg/100g). The results indicated that sesame seed is a good source of essential minerals vital for human nutrition (Deme et al., 2017). Another research was done by supplementing various levels of defatted sesame flour of white cultivar i.e. TH-6 form Pakistan to develop high protein and energy food bars. The results showed that defatted sesame flour of TH-6 contains 385 mg/100g potassium, 7.63 mg/100g sodium, 6.19 mg/100g iron and 20.3 mg/100g calcium (Abbas et al., 2016). According to USDA National Nutrient Database for Standard Reference Library, 100 g edible portion of partially defatted sesame flour contains Ca (150 mg), Mg (362 mg), P (810 mg), Fe (14.30 mg) and Zn (10.70 mg), respectively (USDA, 2018). 4.1.3. Amino acids profile Mean squares for amino acids (essential as well as non-essential) of sesame flours showed significant differences (Table 4.5 and 4.6). Concentrations of isoleucine, leucine, lysine, methionine, phenyl alanine, threonine, tryptophan, valine and histidine were ranged from 1.52±0.02 to 4.34±0.10, 3.86±0.12 to 7.54±0.24, 1.11±0.07 to 3.34±0.09, 1.25±0.04 to 3.47±0.09, 2.24±0.05 to 4.48±0.06, 2.55±0.07 to 4.38±0.12, 0.81±0.01 to 2.57±0.06, 2.55±0.05 to 5.20±0.14 and 1.83±0.05 to 3.10±0.08g/100g, respectively (Table 4.7). Among the different cultivars, maximum concentration (4.34±0.10g/100g) of isoleucine was found in TH-6 followed by TS-5 (3.80±0.04g/100g), TS-3 (3.25±0.09g/100g) and S-122 (3.21±0.12g/100g) whereas the lowest isoleucine was found in Latifi (1.52±0.02g/100g). Regarding, leucine (Table 4.7), TH-6 exhibited the highest value (7.54±0.24g/100g) followed by TS-5 (6.91±0.15g/100g), S-122 (6.67±0.14g/100g) and TS-3 (6.46±0.21g/100g). The lowest value of leucine (3.86±0.12g/100g) was observed in Latifi followed by S-131 (4.76±0.16g/100g). 56 Table 4.5. Mean squares for essential amino acids in sesame flours of different cultivars SOV Df Ile Leu Lys Met Phe Thr Trp Val His Varieties 7 2.441** 4.510** 0.819** 1.574** 1.093** 1.055** 1.0566** 2.273** 0.9299** Error 16 0.015 0.101 0.009 0.011 0.012 0.038 0.0080 0.024 0.0099 Total 23 ** = Highly significant (P<0.01); Df = Degree of freedom His = Histidine Ile = Isoleucine Leu = Leucine Lys = Lysine Met = Methionine Phe = Phenylalanine Thr = Threonine Trp = Tryptophan Val = Valine 57 Table 4.6. Mean squares for non-essential amino acids in sesame flours of different cultivars SOV Df Ala Arg Asp Glu Gly Cys Pro Ser Tyr Varieties 7 8.866** 1.854** 3.570** 13.268** 9.904** 2.191** 1.417** 5.419** 1.883** Error 16 0.014 0.025 0.060 0.325 0.020 0.011 0.010 0.013 0.019 Total 23 ** = Highly significant (P<0.01); Df = Degree of freedom Ala = Alanine Arg = Arginine Asp = Aspartic acid Cys = Cysteine Glu = Glutamic acid Gly = Glycine Pro = Proline Ser = Serine Tyr = Tyrosine 58 Table 4.7. Essential amino acids composition (g/100g) of sesame flours of different cultivars Varieties Ile Leu Lys Met Phen Thr Trp Val His TH-6 4.34±0.10a 7.54±0.24a 3.34±0.09a 3.47±0.09a 4.48±0.06a 4.38±0.12a 2.57±0.06a 5.20±0.14a 3.10±0.08a TS-5 3.80±0.04b 6.91±0.15ab 2.85±0.06b 2.97±0.07b 3.97±0.08b 3.87±0.15ab 2.18±0.12b 4.80±0.10a 2.62±0.02b TS-3 3.25±0.09c 6.46±0.21bc 2.34±0.06bc 2.51±0.04c 3.52±0.08c 3.53±0.08bcd 1.74±0.01c 4.23±0.07b 2.11±0.03cd Til-89 2.78±0.04d 5.92±0.21cd 2.01±0.04cd 1.92±0.05d 3.03±0.06d 3.02±0.10de 1.23±0.02d 3.79±0.05cd 1.76±0.05e S-122 3.21±0.12c 6.67±0.14abc 2.73±0.03b 2.82±0.08b 3.97±0.04b 3.83±0.16ab 2.03±0.04b 4.09±0.12bc 3.24±0.08a S-117 2.72±0.05d 5.23±0.20de 2.54±0.05b 2.33±0.06c 3.43±0.08c 3.39±0.10bc 1.61±0.02c 3.58±0.09d 2.73±0.09b S-131 2.08±0.05e 4.76±0.16e 1.88±0.03d 1.72±0.03d 2.93±0.06d 2.92±0.09e 1.10±0.02d 3.05±0.04e 2.25±0.03c Latifi 1.52±0.02f 3.86±0.12f 1.11±0.07e 1.25±0.04e 2.24±0.05e 2.55±0.07e 0.81±0.01e 2.55±0.05f 1.83±0.05de Means having same letters are statistically non-significant (P>0.05) Means±S.D 59 Results for lysine (Table 4.7) revealed maximum content (3.34±0.09g/100g) in TH-6 whereas the lowest value of lysine (1.11±0.07 g/100g) was noticed in Latifiand S-131 (1.88±0.03g/100g). Means for methionine (Table 4.7) the highest contents (3.47±0.09g/100g) in TH-6, followed by TS-5 (2.97±0.07g/100g) and S-122 (2.73±0.03 g/100g) whereas the lowest methionine (1.25±0.04g/100g) was found in Latifi followed by S-131 (1.72±0.03g/100g). Results for the phenylalanine (Table 4.7) exhibited the values ranged from 2.24±0.05 to 4.48±0.06g/100g. Means for threonine revealed the highest concentration (4.38±0.12g/100g) in TH-6 (white sesame cultivar) and TS-5 (3.87±0.15g/100g). The lowest levels of threonine (2.55±0.07g/100g) were found in Latifi and S-131 (2.92±0.09g/100g). Mean values for tryptophan (Table 4.7) showed the highest values i.e. 2.57±0.06 and 2.18±0.12g/100g in TH-6 and TS-5, respectively whereas the minimum value of tryptophan (0.81±0.01g/100g) was found in Latifi. Results for valine showed in Table 4.7, exhibited the maximum content (5.20±0.14g/100g) in TH-6 followed by TS-5 (4.80±0.10g/100g), TS-3 (4.23±0.07g/100g) andS-122 (4.09±0.12 g/100g) in Til-89. Regarding means for histidine (Table 4.7), the highest value (3.24±0.08g/100g) was observed in S-122 and TH-6 (3.10±0.08 g/100g), whereas, the least amount was in Til-89 (1.76±0.05g/100g) followed by Latifi (1.83±0.05g/100g). Overall, all essential amino acids were significantly higher in white sesame cultivars except histidine which was higher in black sesame. Overall values of alanine arginine, aspartic acid, glutamic acid, glycine, cysteine, proline, serine and tyrosine were ranged from 0.78±0.02 to 5.27±0.14, 1.87±0.08 to 4.83±0.09, 5.87±0.16 to 8.95±0.28, 12.23±0.22 to 18.67±0.20, 1.19±0.02 to 5.96±0.13, 0.57±0.01 to 3.15±0.09, 0.75±0.03 to 3.85±0.07, 0.23±0.01 to 3.90±0.08, and 1.82±0.05 to 4.25±0.07g/100g, respectively (Table 4.8). Among the different cultivars, the highest concentration of alanine (5.27±0.14g/100g) was found in TH-6 followed by TS-5 (4.79±0.09g/100g) and TS-3 (4.25±0.05g/100g) whereas the lowest value in Latifi (0.78±0.02g/100g). Mean values for arginine (Table 4.8) exhibited maximum concentration of arginine (4.83±0.09g/100g) in TH-6 and TS-5 (4.01±0.09g/100g. However, the lowest value of arginine was observed in Latifi (1.87±0.08g/100g) and S-131 (2.24±0.02g/100g). 60 Table 4.8. Non-essential amino acids composition (g/100g) of sesame flours of different cultivars Varieties Ala Arg Asp Glu Gly Cys Pro Ser Tyr TH-6 5.27±0.14a 4.83±0.09a 8.95±0.28a 18.67±0.20a 5.96±0.13a 3.15±0.09a 3.85±0.07a 3.90±0.08a 4.25±0.07a TS-5 4.79±0.09b 4.01±0.09b 8.51±0.13ab 17.13±0.51ab 5.42±0.09b 2.81±0.04b 3.41±0.03b 3.36±0.10b 3.89±0.12b TS-3 4.25±0.05c 3.63±0.06bc 7.89±0.02b 16.54±0.38bc 4.97±0.06c 2.29±0.11c 2.87±0.05c 2.83±0.11c 3.36±0.10c Til-89 3.86±0.07d 3.37±0.12cd 7.03±0.12c 16.07±0.29cd 4.53±0.14d 1.79±0.04d 2.36±0.06d 2.33±0.05d 2.91±0.07d S-122 2.18±0.03e 3.01±0.15d 6.82±0.11c 15.62±0.46bcd 2.82±0.05e 2.23±0.06c 2.28±0.02b 1.42±0.05e 2.86±0.06d S-117 1.86±0.03e 2.85±0.06de 6.76±0.06cd 14.03±0.19de 2.33±0.04f 1.72±0.03d 1.79±0.09c 1.03±0.02f 2.54±0.08de S-131 1.23±0.03f 2.24±0.02e 6.14±0.12de 13.42±0.21e 1.74±0.02g 1.08±0.03e 1.25±0.06d 0.59±0.01g 2.35±0.06e Latifi 0.78±0.02g 1.87±0.08f 5.87±0.16e 12.23±0.22f 1.19±0.02h 0.57±0.01f 0.75±0.03e 0.23±0.01h 1.82±0.05f Means having same letters are statistically non-significant (P>0.05) Means ± S.D 61 Results for aspartic acid (Table 4.8) exhibited the values ranged from 5.87±0.16 to 8.95±0.28 g/100g. Among different cultivars, the maximum content of aspartic acid was obtained in TH- 6 (8.95±0.28g/100g) followed by TS-5 (8.51±0.13g/100g), TS-3 (7.89±0.02g/100g) and (7.03±0.12g/100g) in Til-89. The lowest content of aspartic acid was found in Latifi (5.87±0.16g/100g) followed by S-131 (6.14±0.12g/100g). Means for glutamic acid (Table 4.8) demonstrated the values ranged from 12.23±0.22 to 18.67±0.20 g/100g. Among different cultivars, the maximum concentration of glutamic acid was achieved in TH-6 (18.67±0.20g/100g) followed by TS-5 (17.13±0.51g/100g), TS-3 (16.54±0.38g/100g) and (16.07±0.29g/100g) in Til-89. The minimum concentration of glutamic acid was found in Latifi (12.23±0.22g/100g) followed by S-131 (13.42±0.21g/100g). Results for glycine (Table 4.8) revealed the values ranged from 1.19±0.02 to 5.96±0.13 g/100g. Among various cultivars, the highest value of glycine was found in TH-6 (5.96±0.13g/100g) followed by TS-5 (5.42±0.09g/100g) and TS-3 (4.97±0.06g/100g) whereas the lowest value (1.19±0.02g/100g) was in Latifi and S-131 (1.74±0.02g/100g). Mean values for cysteine (Table 4.8) were ranged from 1.79±0.04 to 3.85±0.07g/100g in white sesame flours and 0.57±0.01 to 2.23±0.06 g/100g in black sesame varieties. The highest cysteine contents (3.15±0.09g/100g) were noted in TH-6 and TS-5 (2.81±0.04g/100g). Means for proline (Table 4.8) were ranged from 0.75±0.03 to 3.85g/100g. Among the different cultivars, TH-6 showed the maximum value (3.85±0.07g/100g) of proline whereas the minimum value of cysteine was observed in Latifi (0.75±0.03g/100g) followed by S-131 (1.25±0.06g/100g). Mean results for serine and tyrosine (Table 4.8) exhibited the values ranged from 0.23±0.01to 3.90±0.08 and 1.82±0.05 to 4.25±0.07g/100g, respectively. Among different cultivars, TH-6 showed the highest concentration (3.90±0.08g/100g and 4.25±0.07g/100g) of these amino acids whereas the lowest concentrations (0.23±0.01g/100g and 1.82±0.05g/100g) were found in Latifi and S-131 (0.59±0.01 and 2.35±0.06g/100g). In a study, amino acid analysis of black and white sesame varieties from Chinese origin revealed significant amount of both essential and non-essential amino acids in both type of cultivars when compared with WHO/FAO standards for both infants and adults except lysine which were insufficient to fulfill the requirements of infants. Furthermore, sulphur containing amino acids like methionine and cysteine were also found in significantly higher quantities than that of WHO standards for both infants and adults. The non-essential amino acids were 62 also higher in these varieties (Radha et al., 2008). In another study, amino acid profile of white and black sesame cultivars exhibited their equal effectiveness for supplementation in low protein flours from cereals for infant feeding. These type of products could be helpful to curtail different disorders related to protein deficiency like Kwashiorkor and Marasmus in children (Elleuch et al., 2007). In another study, sesame-wheat flour cookies were assessed for nutritional composition and sensory parameters. The results revealed presence of essential as well as non-essential amino acids particularly glycine (5.4 g/100g), leucine (6.34 g/100g), methionine (2.91 g/100g), arginine (6.07 g/100g), aspartic acid (10.66 g/100g) and glutamic acid (16.26 g/100g). It was further found that the proportion of amino acids in sesame seed was comparable to those in pumpkin, melon and gourd seeds, and their utilization is suggested to achieve the recommended daily allowance of these amino acids (Olagunju and Ifesan, 2013). 4.1.4. Antioxidant potential Mean squares for antioxidant potential of sesame flours of white and black cultivars showed significant differences with respect to total phenolic contents, DPPH (2, 2-dipheny l-1- picrylhydrazyl) scavenging activity and β-carotene bleaching activity (Table 4.9). Means for total phenolic contents (Table 4.10) exhibited values ranged from 1.56±0.025 to 7.32±0.219mg GAE/g. Among the different cultivars, the maximum value of total phenolic contents was observed in S-122 (7.32±0.22mg GAE/g) followed by S-117 (6.58±0.12 mg GAE/g) and S- 131 (5.69±0.09mg GAE/g). The minimum phenolics were present in Til-89 (1.56±0.03mg GAE/g) and TS-3 (2.69±0.06mg GAE/g). It is obvious from the results that black sesame cultivars were rich in phenolics than that of white cultivars. In a study, sesame extract was analyzed for the total phenolic compounds as gallic acid equivalents/gram of sample. The results revealed that black varieties contain more polyphenols (5.38mg GAE/g) as compared to white ones, which can be extracted and further utilized in pharmaceutical and cosmetics industries (Zheng and Wang, 2001). In another study, Djeridane et al. (2006) found sufficient phenolic compounds (2.88mg GAE/g) in white sesame varieties. They further recommended their utilization to alleviate several human diseases by altering metabolism and reduction in mortality due to the presence of numerous phytochemicals. 63 Table 4.9. Mean squares for antioxidant potential of sesame flours of different cultivars SOV Df Total Phenolic Contents DPPH free radical β -carotene bleaching scavenging activity activity Varieties 7 11.382** 34.395** 42.705** Error 16 0.033 4.528 2.659 Total 23 ** = Highly significant (P<0.01); Df = Degree of freedom 64 Table 4.10. Antioxidant potential of sesame flours of different cultivars Varieties Total phenolic contents DPPH scavenging activity β-carotene bleaching (mg GAE/g) (% inhibition) activity (% inhibition) TH-6 4.86±0.06d 56.55±1.09abc 42.63±1.10abc TS-5 3.72±0.076e 54.34±0.63bc 40.86±0.43bcd TS-3 2.69±0.064f 52.13±0.93c 37.21±0.90de Til-89 1.56±0.025g 51.65±0.98c 35.14±0.95e S-122 7.32±0.219a 61.16±2.13a 46.92±1.34a S-117 6.58±0.115b 59.23±0.80ab 43.63±0.35ab S-131 5.69±0.087c 56.72±1.63abc 41.02±0.56bcd Latifi 4.02±0.075e 53.41±0.92bc 38.37±1.32cde Means having same letters are statistically non-significant (P>0.05) Means ± S.D 65 Mean results for DPPH scavenging activity (Table 4.10) showed the values ranged from 51.65±0.98 to 61.16±2.13% inhibition. S-122 revealed the highest scavenging activity (61.16±2.13% inhibition) followed by S-117 (59.23±0.80%), S-131 (56.72±1.63%) and TH-6 (56.55±1.09 % inhibition). It is apparent from the results that DPPH scavenging activity of black sesame cultivars was slightly higher than that of white varieties. The lowest % inhibition was observed in Til-89 (51.65±0.98%) and TS-3 (52.13±0.93%). Antioxidant activities of sesame extracts were analyzed by using 2, 2-dipheny l-1-picrylhydrazyl (DPPH) methods. The white sesame varieties showed inhibiting against DPPH (upto 56.37%) as compared to standard commercial antioxidants i.e. BHA and TBHQ (Srinivasan, 2005). In a comparative study, sesame cake extracts of both black and white sesame were studied for antioxidants activity using DPPH method. The results revealed higher ability of inhibiting DPPH in black sesame extracts i.e. 61.6%. Moreover, the yield of ethanolic extract of black sesame in powder form was 78.4mg GAE/g (Anilakumar et al., 2010). Means for β-carotene bleaching activity (Table 4.10) revealed the values ranged from 35.14±0.95to 46.92±1.34% inhibition. The results of various sesame cultivars exhibited higher value (46.92±1.34% inhibition) in S-122 followed by S-117 (43.63±0.35% inhibition), and TH-6 (42.63±1.10% inhibition). In a study, white sesame cake extracts of different cultivars were studied. There was strong correlation in phenolic compounds and antioxidants activity. These compounds are widely distributed in plants including oilseeds like sesame seeds. These are natural sources of antioxidants with the potential utilization in array of foods. Further, it was found that sesame seeds exhibit greater antioxidant potential as indicated by β- carotenebleaching assay (Kumar, 2009). In another study, black sesame extracts were examined for antioxidant activity by using β-carotene bleaching method. The results showed comparatively higher inhibiting power in black sesame extracts than that of white onesmainly due to high phenolic compounds in them. This study also revealed that antioxidant activity is further dependent on the extraction solvent, concentration of extract and the assay chosen (Visavadiya et al., 2009). 4.1.5. Bioactive components Mean squares for bioactive components in sesame flours of different cultivars represented significant variances with respect to bioactive components i.e. sesamin, sesamol and sesamolin (Table 4.11). Means for the sesamin, sesamol and sesamolin (Table 4.12) exhibited the values 66 ranged from 1521.00±27.30 to 3996.00±129.90ppm, 3121.00±86.60 to 4303.00±133.37ppm and 1532.33±31.89 to 3564.00±65.82ppm, respectively. Among the different cultivars, the maximum value of sesamin was observed in TH-6 (3996.00±129.90) followed by TS-5 (3753.00±120.67ppm) and TS-3 (3502.00±59.47ppm), whereas, the minimum sesamin concentration (1521.00±27.30ppm) was observed in Latifi and S-131 (1756.00±48.50 ppm). Results revealed that white cultivars of sesame contain more sesamin contents as compared to black cultivars. The results for the sesamol (Table 4.12) showed the highest concentration (4303.00±133.37ppm) of sesamolin S-122 followed by S-117 (4152.00±68.13ppm) and S-131 (3908.00±80.25 ppm) Likewise, sesamolin contents (Table 4.12) were higher in S-122 (3564.00±65.82 ppm) and S-117 (3321.00±119.51 ppm) with the lowest levels in Til-89 (1532.33±31.89 ppm). It is obvious from the results that sesamin and sesamol contents were higher in white sesame cultivars whereas sesamolin was more in black cultivars. In a study, 62 Chinese cultivars of sesame seeds of mixed colors i.e. white, black, and brown were assessed for various bioactive components. The results showed higher concentrations of sesamin and sesamol (3602ppm and 3011ppm) in cultivar Muzhenbai, from Anhui and the cultivar having lowest sesamolin contents (1350ppm) was from Shanxi. It was further reported that sesamin and sesamol levels were higher than those grown in other countries like Thailand (Rangkadilok et al., 2010). Through successful breeding programs, levels of bioactive components can be elevated with more utilization as functional foods, pharmaceutical products and cosmetics (Rao, 2004). Though, seed coat color plays an important role in sesame lignin pathways, but the findings of this instant activity revealed more sesamol and sesamolin contents in black varieties as compared to white ones (Tashiro et al., 1990). However, black sesame cultivars have limited utilization in the development of different food products due to less appreciation for the black color of the end products and presence of some inherent components that may cause slightly bitter taste. 67 Table 4.11. Mean squares for bioactive components in sesame flours of different cultivars SOV Df Sesamin Sesamol Sesamolin Varieties 7 2909137.0** 470153.0** 1762119.0** Error 16 20908.0 34500.0 21177.0 Total 23 ** = Highly significant (P<0.01); Df = Degree of freedom 68 Table 4.12. Bioactive components (ppm) in sesame flours of different cultivars Varieties Sesamin Sesamol Sesamolin TH-6 3996.00±129.90a 3837.00±134.36a-d 2257.00±95.84d TS-5 3753.00±120.67ab 3587.00±130.48cde 1942.00±83.14de TS-3 3502.00±59.47bc 3342.00±98.15de 1778.00±53.12e Til-89 3201.00±105.83c 3121.00±86.60e 1532.33±31.89e S-122 2141.33±70.14d 4303.00±133.37a 3564.00±65.82a S-117 1979.00±39.58d 4152.00±68.13ab 3321.00±119.51ab S-131 1756.00±48.50de 3908.00±80.25abc 3102.00±110.85bc Latifi 1521.00±27.30e 3652.00±104.50bcd 2862.00±75.06c Means having same letters are statistically non-significant (P>0.05) Means ± S.D 69 4.2. Analysis of Sesame Flour Supplemented Doughnuts White and black sesame flour supplemented doughnuts were examined for sensory characteristics e.g. color, aroma, taste, texture, chewability, mouthfeel and overall acceptability by using nine point hedonic score system. 4.2.1. Sensory evaluation Mean values for effect of white and black sesame flour supplementation on sensory evaluation of doughnuts (Table 4.13) indicated that color, aroma, taste, texture, chewability, mouthfeel and overall acceptability differed significantly with respect to varieties (V) and treatments (T) whereas, all quality attributes exhibited non-significant differences regarding interaction between sesame varieties (V) and treatments (T). Means for effect of varying concentrations of sesame flours on color of doughnuts revealed maximum acceptance (7.98±0.16) for TH-6 followed by TS-5 (7.73±0.14) and TS-3 (7.71±0.12) whereas doughnuts made from Latifi (5.66±0.03) and S-131 (5.74±0.05) got the minimum scores (Table 4.14). Overall, doughnuts supplemented with white sesame flours were more appreciated as compared to black ones which have higher levels of black pigments naturally. Regarding supplementation of various levels of both white and black sesame flours, doughnuts having 10g/100g white and black sesame flours (T1) were extremely liked by panelists (7.62±0.15) followed by doughnuts with 20g/100g of sesame flour (7.39±0.12). This tendency was might be due to their close resemblance with other commercially available sesame-based products. In a study, black sesame seed flour was incorporated in leavened pan bread in order to check its impact on physical, nutritional and sensory attributes. The results revealed increase in the dark color of the products with higher levels of supplementation. This was might be due to the natural black pigments present in the outer layers of sesame seeds (Alobo, 2001). Likewise, biscuits having various levels of sunflower protein isolates, exhibited variations in color especially with higher levels of protein isolates. This was also might be due to more protein available for Maillard reaction (Claughton and Pearce, 1989). 70 Table 4.13. Mean squares for sensory evaluation of white and black sesame flour supplemented doughnuts SOV Df Color Aroma Taste Texture Chewability Mouthfeel Overall acceptability Varieties (V) 7 11.879** 14.606** 18.543** 16.860** 13.071** 12.532** 14.358** Treatments 5 164.721** 111.225** 124.058** 134.547** 134.144** 118.965** 134.600** (T) V x T 35 0.061NS 0.004NS 0.011NS 0.003NS 0.008NS 0.085NS 0.005NS Error 432 0.088 0.088 0.105 0.105 0.090 0.111 0.101 Total 479 ** = Highly significant (P<0.01); NS = Non-significant (P>0.05); Df = Degree of freedom 71 Table 4.14. Effect of white and black sesame flour supplementation on the color of doughnuts Treatments Varieties T0 T1 T2 T3 T4 T5 Means TH-6 7.35±0.19 8.72±0.10 8.35±0.12 7.89±0.08 7.72±0.06 7.92±0.09 7.98±0.16a TS-5 7.35±0.19 8.63±0.08 8.30±0.10 7.65±0.07 7.27±0.06 7.21±0.07 7.73±0.14b TS-3 7.35±0.19 8.52±0.07 8.29±0.09 7.53±0.06 7.18±0.05 7.16±0.06 7.71±0.12bc Til-89 7.35±0.19 8.41±0.06 8.27±0.08 7.44±0.07 7.09±0.04 7.12±0.04 7.61±0.11c S-122 7.35±0.19 6.83±0.05 6.69±0.07 5.72±0.08 4.69±0.20 4.31±0.06 5.92±0.09d S-117 7.35±0.19 6.72±0.08 6.54±0.06 5.61±0.06 4.58±0.04 4.22±0.02 5.83±0.07de S-131 7.35±0.19 6.63±0.03 6.42±0.05 5.50±0.05 4.46±0.05 4.13±0.05 5.74±0.05ef Latifi 7.35±0.19 6.56±0.02 6.31±0.04 5.39±0.07 4.35±0.04 4.02±0.04 5.66±0.03f Means 7.35±0.19c 7.62±0.15a 7.39±0.12b 6.59±0.10d 5.91±0.15e 5.70±0.13f Means having same letters are statistically non-significant (P>0.05) Means ± S.D 72 Appearance and color are considered the first attributes among foods to evaluate the quality. It also helps to observe other characteristics like flavor and taste which are strongly associated with the color (Meilgaard et al., 2007). Results for the effect of sesame flours on aroma of doughnuts (Table 4.15) showed that the doughnuts made from TH-6 (8.01±0.26) were liked most by the panelists followed by TS-5 (7.77±0.20) and TS-3 (7.69±0.16) whereas minimum score was observed in Latifi (5.67±0.21) and S-131 (5.76±0.18). It is obvious from the results that doughnuts with white sesame flour were more liked by the panelists than that of black sesame flour supplemented ones. Regarding supplementation levels, means for aroma showed that doughnuts supplemented with 10g/100g of both white and black sesame flour were more liked (7.64±0.18) by the panelists followed by doughnuts containing 20g/100g of sesame flour (7.41±0.16). Sesame seeds and their co-products have typical aroma due to presence of numerous volatile compounds. Doughnut containing 10- 20% defatted sesame flour were even more liked as compared to doughnuts without supplementation. However, higher supplementation levels exhibited intense beany aroma and were not acceptable for the sensor assessors. In an earlier study, it was found that in sesame-based products, aroma is also affected by the appearance and color of the products as these are considered the first quality attributes used for the assessment of the product (Olayanju et al., 2006). Means for the effect of white and black sesame flour supplementation on the taste of doughnuts different concentrations indicated (Table 4.16) that doughnuts made from white sesame cultivar TH-6 found highest concentration (8.03±0.16) followed by TS-5 (7.79±0.14) and TS-3 (7.71±0.12) while the lowest concentration was attained by Latifi (5.68±0.06) and S-131 (5.77±0.07). Again, doughnuts made with white sesame flour were more valued by the panelists due to better palatability whereas doughnut made from various levels of black sesame cultivars got lower scores ranged from 5.68±0.06 to 5.97±0.09. Regarding different supplementation levels, doughnuts containing 10g/100g of white and black sesame flour got maximum acceptance (7.66±0.23) from the panelists followed by doughnuts having 20g/100g of sesame flour (7.43±0.20). 73 Table 4.15. Effect of white and black sesame flour supplementation on the aroma of doughnuts Treatments Varieties T0 T1 T2 T3 T4 T5 Means TH-6 7.36±0.12 8.76±0.09 8.37±0.09 7.91±0.10 7.74±0.06 7.95±0.09 8.01±0.26a TS-5 7.36±0.12 8.65±0.08 8.34±0.06 7.67±0.08 7.30±0.03 7.31±0.07 7.77±0.20b TS-3 7.36±0.12 8.54±0.05 8.31±0.07 7.56±0.07 7.21±0.05 7.19±0.06 7.69±0.16bc Til-89 7.36±0.12 8.43±0.03 8.29±0.08 7.46±0.06 7.11±0.04 7.14±0.05 7.63±0.15c S-122 7.36±0.12 6.85±0.08 6.71±0.10 5.74±0.04 4.71±0.02 4.33±0.04 5.95±0.19d S-117 7.36±0.12 6.74±0.09 6.56±0.07 5.65±0.06 4.62±0.07 4.26±0.03 5.85±0.17de S-131 7.36±0.12 6.67±0.05 6.46±0.05 5.54±0.05 4.50±0.08 4.18±0.02 5.76±0.18ef Latifi 7.36±0.12 6.60±0.07 6.35±0.02 5.43±0.07 4.39±0.09 4.07±0.01 5.67±0.21f Means 7.36±0.12c 7.64±0.18a 7.41±0.16b 6.61±0.14d 5.93±0.11e 5.79±0.09f Means having same letters are statistically non-significant (P>0.05) Means ± S.D 74 Table 4.16. Effect of white and black sesame flour supplementation on the taste of doughnuts Treatments Varieties T0 T1 T2 T3 T4 T5 Means TH-6 7.40±0.27 8.77±0.09 8.39±0.06 7.93±0.10 7.76±0.06 7.97±0.09 8.03±0.16a TS-5 7.40±0.27 8.67±0.08 8.36±0.09 7.69±0.08 7.32±0.05 7.33±0.08 7.79±0.14b TS-3 7.40±0.27 8.56±0.05 8.33±0.07 7.57±0.07 7.23±0.04 7.22±0.07 7.71±0.12bc Til-89 7.40±0.27 8.46±0.03 8.32±0.05 7.48±0.06 7.14±0.03 7.17±0.06 7.66±0.10c S-122 7.40±0.27 6.87±0.08 6.73±0.10 5.76±0.09 4.73±0.07 4.36±0.05 5.97±0.09d S-117 7.40±0.27 6.76±0.06 6.58±0.04 5.63±0.05 4.60±0.06 4.24±0.04 5.86±0.08de S-131 7.40±0.27 6.65±0.05 6.44±0.03 5.52±0.04 4.48±0.05 4.14±0.03 5.77±0.07ef Latifi 7.40±0.27 6.58±0.07 6.33±0.02 5.41±0.03 4.37±0.04 4.04±0.02 5.68±0.06f Means 7.40±0.27c 7.66±0.23a 7.43±0.20b 6.62±0.18d 5.93±0.16e 5.79±0.13f Means having same letters are statistically non-significant (P>0.05) Means ± S.D 75 There was slight bitterness in the taste of end products containing higher levels of sesame flours from black cultivars. In a study, biscuits were developed using defatted sesame flour. The results regarding consumer acceptability showed gradual decrease in the acceptance of products due to slight bitter taste linked with the presence of some inherent compounds (Olagunju and Ifesan, 2013). Texture is considered as a major quality feature that is directly linked with the freshness of product. It is defined as “the sum of cutaneous and kinesthetic sensations resulted from oral and manual manipulation. It covers residual and masticatory properties, mouthfeel, visual and auditory attribute of a food (Costell and Duran, 2002). Means for the impact of sesame flour supplementation on the texture of doughnuts exhibited significant difference among the treatment due to variations in the white and black cultivars. It is apparent from the results (Table 4.17) that overall texture of doughnuts containing sesame flour of white cultivars i.e.TH-6 (8.04±0.28), TS- 5 (7.81±0.23), TS-3 (7.73±0.18) and Til-89 (7.68±0.15) was more appreciated by the panelists. Concerning different supplementation levels of both white and black sesame flours for the development of doughnuts, doughnuts containing 10-20 g/100g of sesame flour i.e.T1 (7.68±0.06) and T2 (7.45±0.05) got more acceptability due to better texture. The addition of defatted sesame flour improved the texture of product by imparting fine pore size which were collapsed with higher supplementation levels. In a similar type of study, organoleptic and nutritional assessment of biscuits developed from sesame revealed significant decrease in the score of texture as the sesame flour supplementation was increased (Gandhi and Taimini, 2009). Likewise, the results for nutritional and sensory evaluation of bread fortified with defatted soybean fours and sesame meals showed decrease in the texture with higher levels of supplementation (Serna et al., 1999). Means for the influence of white and black sesame flour supplementation on the chewability of doughnuts indicated maximum acceptance of doughnuts developed with white sesame cultivar TH-6 (8.06±0.31) followed by TS-5 (7.82±0.28) and TS-3 (7.75±0.23) whereas doughnuts having various levels of sesame flour of black cultivars i.e. Latifi (5.72±0.11) and S-133 (5.83±0.13) got the minimum acceptance. Regarding supplementation of both white and black sesame flours for the production of high protein doughnuts, products with lower levels of supplement (10-20%) exhibited better chewability (7.70±0.16 and 7.47±0.15, respectively). 76 Table 4.17. Effect of white and black sesame flour supplementation on the texture of doughnuts Treatments Varieties T0 T1 T2 T3 T4 T5 Means TH-6 7.38±0.24 8.79±0.09 8.40±0.10 7.95±0.07 7.78±0.06 7.99±0.09 8.04±0.28a TS-5 7.38±0.24 8.69±0.08 8.37±0.09 7.71±0.08 7.34±0.03 7.37±0.07 7.81±0.23b TS-3 7.38±0.24 8.59±0.07 8.36±0.08 7.59±0.06 7.25±0.05 7.24±0.06 7.73±0.18bc Til-89 7.38±0.24 8.48±0.05 8.34±0.07 7.50±0.05 7.18±0.04 7.19±0.05 7.68±0.15c S-122 7.38±0.24 6.89±0.06 6.75±0.06 5.79±0.04 4.75±0.07 4.39±0.03 5.99±0.13d S-117 7.38±0.24 6.79±0.05 6.61±0.05 5.66±0.06 4.63±0.04 4.27±0.02 5.88±0.11de S-131 7.38±0.24 6.68±0.04 6.47±0.04 5.54±0.05 4.51±0.03 4.16±0.01 5.81±0.10ef Latifi 7.38±0.24 6.60±0.03 6.35±0.02 5.43±0.03 4.39±0.02 4.07±0.02 5.70±0.08f Means 7.38±0.24c 7.68±0.06a 7.45±0.05b 6.64±0.06d 5.95±0.05e 5.81±0.06f Means having same letters are statistically non-significant (P>0.05) Means ± S.D 77 Table 4.18. Effect of white and black sesame flour supplementation on the chewability of doughnuts Treatments Varieties T0 T1 T2 T3 T4 T5 Means TH-6 7.41±0.10 8.81±0.09 8.42±0.10 7.97±0.11 7.80±0.12 7.81±0.09 8.06±0.31a TS-5 7.41±0.10 8.71±0.08 8.39±0.09 7.73±0.08 7.36±0.10 7.39±0.07 7.82±0.28b TS-3 7.41±0.10 8.61±0.05 8.38±0.07 7.61±0.07 7.27±0.08 7.26±0.06 7.75±0.23bc Til-89 7.41±0.10 8.50±0.03 8.36±0.06 7.52±0.06 7.20±0.07 7.21±0.05 7.70±0.21c S-122 7.41±0.10 6.91±0.08 6.77±0.05 5.81±0.05 4.77±0.06 4.41±0.04 6.01±0.19d S-117 7.41±0.10 6.81±0.06 6.63±0.04 5.68±0.04 4.65±0.04 4.29±0.03 5.90±0.16de S-131 7.41±0.10 6.71±0.05 6.49±0.03 5.56±0.03 4.53±0.03 4.18±0.02 5.83±0.13ef Latifi 7.41±0.10 6.62±0.03 6.37±0.02 5.45±0.02 4.41±0.02 4.09±0.01 5.72±0.11f Means 7.41±0.19c 7.70±0.16a 7.47±0.15b 6.66±0.13d 5.97±0.12e 5.83±0.10f Means having same letters are statistically non-significant (P>0.05) Means ± S.D 78 The doughnut with higher levels of sesame four were slight difficult to chew as reflected from the rubbery texture. This was might be due to higher levels of protein which after frying of the product turned into elastic material. The score for the chewability of bread samples was decreased as the supplementation level of soy flour was increased (Ndife et al., 2011). Means for the effect of white and black sesame flour supplementation on the mouthfeel of doughnuts are expressed in Table 4.19. It is apparent from the results that doughnuts prepared from TH-6 (8.05±0.32) followed by TS-5 (7.84±0.29) and TS-3 (7.77±0.27) showed maximum acceptance due to better mouthfeel whereas doughnuts containing sesame flour of cultivars Latifi (5.78±0.11) and S-133 (5.89±0.14) got minimum acceptance. Regarding different combinations of both white and black sesame for product development, doughnuts containing 10g/100g of sesame flour (7.73±0.19) and 20g/100g of sesame flour (7.49±0.16) were most liked by the panelists. The score for the mouthfeel decreased as the level of sesame flour supplementation was increased. This was might be due to better texture, aroma and appearance of the doughnut containing white sesame flour which ultimately have impact on the overall impression of the respective product. In a similar study, it was found that decrease in the mouthfeel of defatted soy flour supplemented bread was might be due to various baking conditions i.e. time and temperature variables, the state of the dough components, such as fiber, protein and starch whether damaged or undamaged and the extents of absorbed water during dough mixing which contribute to the final mouthfeel of the bread (Serrem et al., 2011). Results for the effect of white and black sesame flour supplementation (Table 4.20) on the overall acceptability of doughnuts showed that the maximum score (8.02±0.24) was achieved by the doughnuts made from TH-6 followed by TS-5 (7.79±0.18) and TS-3 (7.72±0.20) while Latifi (5.70±0.10) and S-133 (5.78±0.16) attained the lowest scores (Figure 4.1). The quality of the sesame flour depends on numerous factors such as variety, moisture content and region of production etc. Utilization of sesame in food products enhances their acceptability up to certain levels of supplementation. It is obvious from the results that doughnuts having white sesame flour were more liked by panelists as compared to black ones due to better appearance, aroma, texture, chewiness, mouthfeel and overall acceptability. This was might be due to indigenous likeness for the white sesame in this part of the world and presence of some intrinsic compounds in black sesame seeds that may cause slight bitterness in taste and blackish appearance of the product. 79 Table 4.19. Effect of white and black sesame flour supplementation on the mouthfeel of doughnuts Treatments Varieties T0 T1 T2 T3 T4 T5 Means TH-6 7.39±0.21 8.83±0.09 8.44±0.10 7.99±0.08 7.82±0.06 7.83±0.09 8.05±0.32a TS-5 7.39±0.21 8.73±0.08 8.41±0.09 7.75±0.07 7.38±0.05 7.41±0.07 7.84±0.29b TS-3 7.39±0.21 8.63±0.07 8.40±0.08 7.63±0.06 7.29±0.04 7.28±0.06 7.77±0.27bc Til-89 7.39±0.21 8.53±0.06 8.38±0.07 7.54±0.05 7.22±0.03 7.23±0.05 7.71±0.24c S-122 7.39±0.21 6.93±0.05 6.79±0.06 5.83±0.04 4.79±0.02 4.43±0.04 6.02±0.19d S-117 7.39±0.21 6.83±0.04 6.65±0.05 5.70±0.03 4.67±0.04 4.31±0.02 5.90±0.17de S-131 7.39±0.21 6.73±0.03 6.51±0.04 5.58±0.02 4.55±0.05 4.20±0.03 5.89±0.14ef Latifi 7.39±0.21 6.64±0.02 6.39±0.02 5.47±0.01 4.43±0.03 4.11±0.01 5.78±0.11f Means 7.39±0.21c 7.73±0.19a 7.49±0.16b 6.68±0.13d 5.99±0.11e 5.85±0.09f Means having same letters are statistically non-significant (P>0.05) Means ± S.D 80 Table 4.20. Effect of white and black sesame flour supplementation on the overall acceptability of doughnuts Treatments Varieties T0 T1 T2 T3 T4 T5 Means TH-6 7.38±0.26 8.78±0.07 8.39±0.12 7.93±0.10 7.77±0.12 7.91±0.10 8.02±0.24a TS-5 7.38±0.26 8.68±0.08 8.36±0.09 7.70±0.08 7.32±0.10 7.33±0.07 7.79±0.18b TS-3 7.38±0.26 8.57±0.05 8.34±0.08 7.58±0.07 7.23±0.09 7.22±0.06 7.72±0.20bc Til-89 7.38±0.26 8.46±0.03 8.32±0.07 7.49±0.06 7.15±0.08 7.17±0.05 7.66±0.14c S-122 7.38±0.26 6.88±0.08 6.63±0.06 5.83±0.05 4.74±0.06 4.37±0.06 5.97±0.12d S-117 7.38±0.26 6.77±0.06 6.59±0.05 5.65±0.04 4.62±0.04 4.20±0.02 5.86±0.08de S-131 7.38±0.26 6.66±0.05 6.46±0.04 5.54±0.03 4.50±0.03 4.16±0.05 5.78±0.16ef Latifi 7.38±0.26 6.60±0.04 6.35±0.03 5.44±0.02 4.39±0.02 4.06±0.04 5.70±0.10f Means 7.38±0.26c 7.67±0.24a 7.43±0.18b 6.64±0.14d 5.96±0.11e 5.80±0.08f Means having same letters are statistically non-significant (P>0.05) Means ± S.D 81 Regarding various levels of supplementation, doughnuts containing 10g/100g (7.67±0.24) to 20 g/100g (7.43±0.18) of either white or black sesame flours were more acceptable due to delicious nutty aroma & flavor of sesame, mild brownish appearance, uniform texture, nutty mouthfeel and overall impression of the end product. With higher levels of supplementation, the color of doughnut become dark brown alongwith unpleasant nutty aroma, rubbery chewiness and non- uniform mouthfeel due to large pore size. The appearance of popcorn-like aroma and onion-like odor in sesame-based products with higher concentration of sesame is associated with the furylpyrazines. Additionally, the presence of seed coat can also influence the flavor of sesame (Bedigian, 2010). In current study, weaker sweet and nutty flavor of white cultivars was similar to decorticated sesame seeds. Hence, doughnut having sesame flour of these cultivars were more liked by the panelists. In a study, there was decrease in the sensory scores with increased levels of sesame flour used for the preparation of biscuits (Agu and Nididiamaka, 2014). Moreover, the analysis of quality characteristics of sesame-based products revealed that uniform pore size and distribution of constituents have impact on the chewability and mouthfeel characteristics of end- product (Elleuch et al., 2007). Sensory evaluation is a scientific approach to score sensory attributes of food by human senses i.e. taste, smell, touch, vision and hearing (Meilgaard et al., 2007). Furthermore, consumer behavior about food stuff is determined by doing various types of acceptance tests. 4.3. Selection of Best Sesame Cultivar Based on consumer acceptability and better-quality attributes, TH-6 (white sesame cultivar) was selected for further development of sesame flour supplemented doughnuts. Regarding supplementation levels, sesame-based doughnuts having 10g/100g and 20g/100g of sesame flour were considered best. Best selected formulations along with control were analyzed for proximate composition, mineral contents, texture, color, and calorific value. Storage study was also carried out to analyze water activity, peroxide value, thiobarbituric acid number and mold growth. Furthermore, these compositions were further assessed for protein quality through biological evaluation using Sprague Dawly to find out the nutritional and serum profile of school aged children. 82 Black sesame White sesame 9 8 7 6 5 Overall acceptability Overall 4 3 T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5 V1 V2 V3 V4 Figure 4.1. Effect of varieties and treatments on overall acceptability of white and black sesame flour supplemented doughnuts V1= TH-6 (White), S-122 (Black) V2= TS-5 (White), S-117 (Black) V3= TS-3 (White), S-131 (Black) V4= Til-89 (White), Latifi (Black) 83 4.4. Analysis of Selected Sesame Flour Supplemented Doughnuts 4.4.1. Proximate composition Mean values for proximate analysis of sesame flour supplemented doughnuts exhibited significant variations among the treatments (Table 4.21). Means for moisture content (Table 4.22) showed values ranged from 7.53±0.19 to 8.38±0.12%. The highest value (8.38±0.12%) was observed in doughnuts without supplementation followed by doughnuts containing 10g/100g of sesame flour (7.65±0.28%), whereas the lowest moisture (7.53±0.19%) was found in doughnuts with 20g/100g of sesame flour. Results reveal that there was slight decrease in moisture content with increased supplementation of sesame flour. Results for protein content (Table 4.22) revealed values ranged from 10.20±0.26 to 24.85±0.40%. The maximum value (24.85±0.40%) was found in doughnuts developed with 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (22.69±0.31%). It is obvious from the results that there was gradual increase in crude protein contents with increasing the level of sesame flour supplementation from 10 to 20g/100g may be due to high initial protein content (38.97±0.28%) of the defatted sesame flours. In a similar study, sesame-based composite bread samples showed an increase in the protein content from 103.3 to 14.8% with sesame flour supplementation as compared to the control bread (9.2%). This was because of lower protein content of wheat flour than that of sesame flour (Kanu, 2011). Means for crude fat content of doughnuts (Table 4.22) indicated values ranged from 9.25±0.23 to 14.41±0.46%. The maximum value (14.41±0.46%) was confirmed in doughnuts having 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (12.62±0.35%). The fat content of simple doughnuts was 9.25±0.23%. In a similar study, oilseeds (Sesamum indicum) were added in wheat flour and assessed for impact on nutritional composition of leavened pan bread. The fat content of the supplemented products was increased from 5.8 to 10.0% as compared to samples without supplementation (3.7%). The results indicated increase in fat content of the end products due to the presence of higher levels of inherent fat contents in sesame flour (Kanu, 2011). 84 Table 4.21. Mean squares for proximate composition of sesame flour supplemented doughnuts SOV Df Moisture Crude protein Crude fat Crude fiber Ash NFE Treatments 2 2.3569** 4.889** 3.610** 2.839** 2.712** 27.658** Error 6 0.1716 0.381 0.44 0.039 0.143 2.259 Total 8 ** = Highly significant (P<0.01); Df = Degree of freedom 85 Table 4.22. Effect of treatments on the proximate composition (%) of sesame flour supplemented doughnuts Treatments Moisture Protein Fat Fiber Ash NFE T0 8.38±0.12a 10.20±0.26c 9.25±0.23c 1.52±0.08c 0.90±0.11c 68.45±0.90a T1 7.65±0.28b 22.69±0.31b 12.62±0.35b 1.85±0.24b 1.40±0.24b 53.79±0.72b T2 7.53±0.19c 24.85±0.40a 14.41±0.46a 2.27±0.33a 1.84±0.36a 49.10±1.45c Means having same letters are statistically non-significant (P>0.05) Means ± S.D T0= Doughnuts without supplementation act as control T1= Doughnuts supplemented with 10g/100g of sesame flour T2= Doughnuts supplemented with 20g/100g of sesame flour 86 Crude fiber content (Table 4.22) values were ranged from 1.52±0.08 to 2.27±0.33%. The highest value (2.27±0.33%) was revealed in doughnuts developed with 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (1.85±0.24%). This was might be due to higher crude fiber contents of sesame flours (5.84±0.16%) than that of wheat flour. Results for the ash content (Table 4.22) of sesame flour supplemented doughnuts exhibited values ranged from 0.90±0.11 to 1.84±0.36%. The maximum minerals (1.84±0.36%) were observed in doughnuts developed with 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (1.40±0.24%), whereas the minimum value (0.90±0.11%) was in doughnuts without any supplementation. There was gradual increase in ash content with subsequent increase of the sesame flour supplementation which has high ash content (9.93±0.18%) as compared to wheat flour. Similar increase in crude fiber and mineral contents were noted in composite bread samples with increase in the supplementation of sesame flour which has high content of lignin, hemicelluloses and cellulose (Mepba et al., 2007) Means for the NFE (Table 4.22) revealed values ranged from 49.10±1.45 to 68.45±0.90%. The highest value (68.45±0.90%) was observed in doughnuts without supplementation followed by doughnuts containing 10g/100g of sesame flour (53.79±0.72%) and 20g/100g of sesame flour (49.10±1.45%). The decrease in NFE was due to the difference in the proximate composition of the sesame-based doughnuts contributed by the variations in raw materials for moisture, crude protein, crude fat, crude fiber and ash contents. Similarly, supplementation of wheat flour with sesame flour showed reduction in NFE of the composite bread samples. The highest NFE was observed in the control sample (53.90%) whereas the lowest (32.46%) in sample containing sesame flour. This revealed that wheat flour was the main contributor of the NFE in the bread (Mepba et al., 2007). The results are in conformance with a similar study reported that the increased supplementation of wheat flour with sesame flour considerably affected the chemical composition of composite bread. Generally, sesame flour possesses higher levels of crude protein, fat, fiber and ash contentsas compared to wheat flour (Makinde and Akinoso, 2013). In another investigation, sesame flour supplemented bars were developed by adding various levels of defatted sesame flour to increase the protein levels. The results showed decrease in moisture content (5.635 to 3.651%) and NFE (46.04 to 49.17%) whereas increase in crude protein (33.69 to 35.57%), crude fat (0.64 to 0.97%, crude fiber (1.93 to 2.54%) and total ash (2.67 to 3.43%), respectively (Abbas et al., 2016). 87 4.4.2. Mineral profile Results for mineral profile of sesame flour supplemented doughnuts indicated significant differences among the treatments with respect to sodium, potassium, calcium, magnesium, phosphorus, iron and zinc (Table 4.23). Sodium content (Table 4.24) values were ranged from 42.03±0.95 to 65.35±0.74 mg/100g. The highest value (65.35±0.74mg/100g) sodium was in doughnuts having 20g/100g of sesame flour followed by doughnuts containing 10g/100g (56.24±0.43mg/100g), whereas the lowest value (42.03±0.95mg/100g) was noted in doughnuts without supplementation. Sodium imparts a significant role in the fluid balance, transmission of nerve impulse and contraction of muscles inside the body. It performs this function by acting as a part of sodium-potassium pump in the plasma membrane of cells. It also acts as an electrolyte when dissolved in solution and conduct electricity. The 86.33mg/100g sodium content of wheat- sesame bread may provide 5.76% of the RDI of the mineral for both males and females, based on an RDA of 1500 mg/day. Sodium has maximum permitted limit of 2400mg; hence, the consumption of sesame supplemented doughnuts is expected to be useful for health (Deman et al., 2018). Potassium content of doughnuts (Table 4.24) showed the values ranged from 19.65±0.46 to 32.52±0.71 mg/100g. The maximum value (32.52±0.71 mg/100g) was noticed in doughnuts having 20g/100g of sesame flour followed by doughnuts with 10g/100g of sesame flour (24.62±0.28mg/100g). There was increase in the potassium content of doughnuts with increase in the concentration of sesame flour, which has 67.32±1.01 mg/100g potassium. Potassium is an important electrolyte for maintaining the fluid concentration inside body. It also influences the contraction of both smooth and skeletal muscles and heart function and extremely affects the transmission of nerve impulses. RDA of the potassium is 4700mg/day which means (Kiralan et al., 2010). 88 Table 4.23. Mean squares for mineral contents of sesame flour supplemented doughnuts SOV Df Sodium Potassium Calcium Magnesium Phosphorus Iron Zinc (Na) (K) (Ca) (Mg) (P) (Fe) (Zn) Treatments 2 11.338* 6.7561* 18511** 4949.7** 13894** 1.4277** 95.532** Error 6 1.662 0.7924 13 33.6 346 0.0082 0.056 Total 8 * = Significant (P<0.05); ** = Highly significant (P<0.01); Df = Degree of freedom 89 Table 4.24. Effect of treatments on the mineral contents (mg/100g) of sesame flour supplemented doughnuts Treatments Sodium Potassium Calcium Magnesium Phosphorus Iron Zinc (Na) (K) (Ca) (Mg) (P) (Fe) (Zn) T0 42.03±0.95c 19.65±0.46c 84.26±1.70c 95.06±2.31c 385.27±7.78c 10.25±0.15c 10.15±0.23c T1 56.24±0.43b 24.62±0.28b 126.33±0.95b 132.37±4.32b 468.32±14.41b 20.55±0.85b 18.43±0.43b T2 65.35±0.74a 32.52±0.71a 236.38±3.00a 176.21±3.10a 520.18±8.80a 22.64±0.64a 20.92±0.24a Means having same letters are statistically non-significant (P>0.05) Means ± S.D 90 Calcium content (Table 4.24) indicated the values ranged from 84.26±1.70 to 236.38±3.00 mg/100g. The highest value (236.38±3.00 mg/100g) was found in doughnuts with 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (126.33±0.95mg/100g) whereas the lowest value (84.26±1.70mg/100g) was explained in doughnuts without supplementation. Calcium plays a significant role in bone mineralization, hydrolysis of phospholipids in cell, neurotransmitters, phospholipids and activation or deactivation of enzymes through phosphorylation. Intake of calcium fortified diets can help to curtail with certain cancers. It is also important in maintaining the fluid electrolyte balance in the body. An adequate intake of calcium more than 800mg/day is used to decrease the chances of colon cancer. Adults of age 19- 50years require 1000mg/day of calcium. Women and men of age above 50years should increase their intake of calcium to 1200mg/day (Potter and Joseph, 1995). Magnesium content of doughnuts (Table 4.24) revealed the values ranged from 95.06±2.31 to 176.21±3.10mg/100g. The maximum value (176.21±3.10/100g) was found in doughnuts with 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (132.37±4.32mg/100g), whereas the minimum value (95.06±2.31mg/100g) was obtained in doughnuts without supplementation. Overall, magnesium levels were increased as a result of supplementation levels. Magnesium is involved in glycolysis and oxidative phosphorylation. It also contributes to the structural development of bones as well as protein and nucleic acid synthesis, contraction of cardiac and smooth muscles, insulin action and transcriptionof DNA and RNA. An adult has 420mg recommended dietary allowance for Mg (Whitney et al., 2002). The consumption of sesame supplemented doughnuts can easily provide about 25-35% RDA of magnesium. Phosphorus contents (Table 4.24) were ranged from 468.32±14.41 to 520.18±8.80 mg/100g in the intervention treatments. The highest value (520.18±8.80mg/100g) was documented in doughnuts containing 20g/100g of sesame flour whereas doughnuts without supplementation exhibited the lowest value (385.27±7.78mg/100g). The results are in conformance with the study which revealed that phosphorus content was predominantly high in all wheat-sesame based composite breads. Phosphorus is of prime importance in the formation of nucleic acids, development of skeletal tissues, energy transfer and storage and structural roles i.e., acid-base balance of cells and formation of cell membranes. The recommended daily allowance of phosphorus is 700mg/day for both males and females (Awan, 2011). 91 Iron content (Table 4.24) were ranged from 10.25±0.15 to 22.64±0.64 mg/100g in all the treatments. The maximum iron (22.64±0.64 mg/100g) was observed in doughnuts with 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (20.55±0.85mg/100g), whereas the minimum value (10.25±0.15mg/100g) was noted in doughnuts without supplementation. Overall, increased supplementation level of sesame flour increased the iron content due to presence of higher iron content (7.23±0.14 mg/100) in it. Iron is an essential component of hemoglobin also performs an important role in transfer of oxygen from the lungs to the tissues. It also acts as a constituent of myoglobin which also supports metabolism. Iron is also important for development, growth, synthesis of some hormones, connective tissues and normal cellular functioning. The RDA of iron is 0.27-27mg/day (Vaclavik and Christian, 2014). Sesame flour-based doughnuts have the potential to meet almost 100% of the iron requirements of some age groups. Means for the zinc contents of doughnuts are presented in Table 4.24. It is obvious from the results that the highest value (20.92±0.24mg/100g) was noted in doughnuts developed with 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (18.43±0.43mg/100g). Zinc plays an important role in the immune function, synthesis of proteins, wound healing, cell division, synthesis of DNA and cellular metabolism. It also plays an important role for the growth and development during childhood, adolescence and pregnancy. The RDA of zinc is 2-12mg. In earlier studies, significant increase in mineral contents of the sesame-wheat composite bread has been reported (Iombor and Girgih, 2016). 4.4.3. Texture analysis Sesame flour supplemented doughnuts were analyzed for hardness, cohesiveness, springiness and chewiness. Statistical analysis for texture analysis of sesame flour supplemented doughnuts showed significant differences among the doughnuts with respect to hardness, cohesiveness, springiness and chewiness (Table 4.25). 92 Table 4.25. Mean squares for texture analysis of sesame flour supplemented doughnuts SOV Df Hardness Cohesiveness Springiness Chewiness Treatments 2 4438.7** 0.00790** 0.00262* 0.04410** Error 6 16.7 0.000095 0.00032 0.00047 Total 8 * = Significant (P<0.05); ** = Highly significant (P<0.01); Df = Degree of freedom 93 Means for the hardness (Table 4.26) demonstrated values ranged from 42.27±0.81 to 113.72±3.64 N. The maximum value (113.72±3.64N) was found in doughnuts having 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (102.69±1.67N). Hardness is one of the most important attribute of doughnuts texture. It is the force required for first compression of the product. There is a strong relationship between hardness and bulk density. In sesame-based snacks as the level of supplementation was increased, hardness and bulk density of product were also increased (Altan et al., 2008). In another exploration, textural and sensorial attributes of oilseeds-based snacks were studied after the addition of buckwheat in it. The findings of this research revealed increase in the hardness of snacks due to the presence and formation of more complexes between protein and starch in buckwheat (Wojtowicz et al., 2013). Similarly, there was increase in the hardness of sesame-based bread with elevated contents of sesame flour (Shittu et al., 2007). Cohesiveness revealed the values ranged from 0.15±0.21 to 0.25±0.42 (Table 4.26). The maximum value (0.25±0.42) was observed in doughnuts having 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (0.22±0.34), whereas the minimum value (0.15±0.21) was in doughnuts without supplementation. Cohesiveness indicates that how well a product resists the second deformation relative to first deformation. Hardness is just because of the more cohesiveness of the product between different molecules. Resultantly, protein binds with starch so increased content of protein resulted in increased cohesiveness of the sesame supplemented products. In a previous study, increase in cohesiveness was accredited to starch expansion and gelatinization as well as share of protein rich ingredients (Ratanatriwong and Barringer, 2007; Sirrichokworrakit et al., 2016). Means for the springiness (Table 4.26) expressed values ranged from 0.71±0.26 to 0.77±0.41. The highest value (0.77±0.41) was noted in doughnuts without supplementation followed by doughnuts containing 10g/100g of sesame flour (0.74±0.36) and doughnuts with 20g/100g of sesame flour (0.71±0.26). Springiness indicates that how well a food product springs back after its first deformation during the first compression (Bourne, 2002). Springiness was decreased with the increase of sesame flour supplementation because higher protein contents cause strong binding of all other ingredients inside the product which leads to the firm and uniform behavior of doughnuts so the hardness in the product increased. 94 Table 4.26. Effect of treatments on the textural characteristics of sesame flour supplemented doughnuts Treatments Hardness Cohesiveness Springiness Chewiness T0 42.27±0.81c 0.15±0.21c 0.77±0.41a 0.23±0.18c T1 102.69±1.67b 0.22±0.34b 0.74±0.36ab 0.32±0.06b T2 113.72±3.64a 0.25±0.42a 0.71±0.26b 0.47±0.02a Means having same letters are statistically non-significant (P>0.05) Means ± S.D 95 Springiness is conversely related to hardness and consequently it decreases due to low moisture and fat content in the product (Shih et al., 2001). Means for the chewiness (Table 4.26) revealed the values ranged from 0.23±0.18 to 0.47±0.02. The maximum value (0.47±0.02) was observed in doughnuts having 20g/100g of sesame flour followed by doughnuts containing 10g/100g of sesame flour (0.32±0.06). Chewiness is also related to hardness as indicated by the definition of cohesiveness (Bourne, 2002). Another research was conducted to evaluate the rheological and sensory attributes of the bread. The results revealed increase in chewiness due to the lower elasticity, viscosity, moisture contents and dough temperatures which resulted in a softer texture (Lee et al., 2007; Rehman et al., 2007). 4.4.4. Color analysis and calorific value Sesame flour supplemented doughnuts were evaluated for L*, a*, b* and calorific values. Mean squares exhibited significant differences among the doughnuts for these attributes (Table 4.27). Means for the L* value of doughnuts (Table 4.28) showed the values ranged from 46.58±0.76 to 55.91±1.13. The maximum value (55.91±1.13) was observed in doughnuts without supplementation followed by doughnuts containing 10g/100g of sesame flour (48.26±0.67), 20g/100g of sesame flour (46.58±0.76). The results indicated that with the increase of sesame flour supplementation there was gradual decrease in the L* value. This was might be due to more chances of Maillard reaction as a result of more available amino acids and protein for reaction with sugars (Budryn et al., 2013). Means for a* value (Table 4.28) unveiled the values ranged from 9.56±0.21 to 15.68±0.31. The maximum value (15.68±0.31) was noted in doughnuts without supplementation followed by doughnuts containing 10g/100g (11.23±0.18) and 20g/100g of sesame flour (9.56±0.21). The results showed the positive values of means which reflect more reddish color rather than greenish one. Though, the values were not extraordinarily high thus awarding least impact of the green or red pigments in the final product. In a previous study, more reddish color (a* value) of fortified breads with anthocyanin rich black rice was due to pigments and high baking temperatures (Xiaonan and Weibiao, 2016). Means for b* value (Table 4.28) demonstrated the values ranged from 18.50±0.25 to 36.42±0.93. The maximum value (36.42±0.93) was noticed in doughnuts without supplementation followed by doughnuts containing 10g/100g (20.69±0.40), 20g/100g of sesame flour (18.50±0.25).The b* values were positive which means that these products are more yellowish in color. High sugar contents initiate the Maillard reaction that resulted in brown color of the final product. 96 Table 4.27. Mean squares for color characteristics and calorific value of sesame flour supplemented doughnuts SOV Df L* a* b* Calorific value Treatments 2 6.0573** 33.657* 40.060** 1447.4* Error 6 0.0559 6.513 2.985 136.4 Total 8 * = Significant (P<0.05); ** = Highly significant (P<0.01); Df = Degree of freedom 97 Table 4.28. Effect of treatments on the color and calorific value of sesame flour supplemented doughnuts Treatments L* a* b* Calorific value (Kcal/100g) T0 55.91±1.13a 15.68±0.31a 36.42±0.93a 350.07±3.47b T1 48.26±0.67b 11.23±0.18b 20.69±0.40b 371.87±8.59ab T2 46.58±0.76b 9.56±0.21c 18.50±0.25c 394.00±7.12a Means having same letters are statistically non-significant (P>0.05) Means ± S.D 98 Equivalent changes in a* and b* values were reported by researchers due to the Maillard reaction and development of dark color in the product (Paraman et al., 2012). In an earlier study, positiveb* value of products was attributed to the presence of more starch contents in the end products (Altan et al., 2008). Means for calorific content of sesame flour supplemented doughnuts (Table 4.28) showed values ranged from 350.07±3.47 to 394.00±7.12 Kcal/100g. The highest value (394.00±7.12Kcal/100g) was noticed in doughnuts with 20g/100g of sesame flour whereas the lowest value (350.07±3.47Kcal/100g) was attained by doughnuts without supplementation. Overall, in the treatments with higher levels of supplementation calorific values were slightly higher which was might be due to more fat content of these products. Similar findings have been reported in earlier studies in which oilseeds-based gluten free crackers and sesame-based products contain 320- 430Kcal/100g (Mohamed et al., 2007). In cashew nut bars, the calorific values were ranged from 377-404 Kcal/100g (Mourao et al., 2009) whereas in banana bars values were 284.00 Kcal/100g (Moura et al., 2013). The calorific value sesame flour supplemented breads ranged from 320 to 390Kcal/100g, depending upon the levels of supplemented sesame flours (Makinde and Akinoso, 2013). 4.4.6. Storage study Mean squares for water activity, peroxide value, thiobarbituric acid number, mold growth of doughnuts (Table 4.29) revealed significant differences among the treatments during the storage. However, the interactions between the treatments and storage intervals non-significant differences. 4.4.6.1. Water activity Means for the water activity (Table 4.30) exhibited the highest value (0.707±0.38) in doughnuts without supplementation followed by doughnuts containing 10g/100g (0.638±0.22) and 20g/100g of sesame flour (0.544±0.16). It was noted that water activity was decreased subsequently with increase in the supplementation level of sesame flour. Storage also has significant effect on the water activity. At the start of study, it was (0.597±0.27) which was steadily increased to 0.635±0.32 and 0.657±0.45 at the mid and end of study, respectively. 99 Table 4.29. Mean squares for the water activity, peroxide value, thiobarbituric acid number and mold growth of sesame flour supplemented doughnuts SOV Df Water activity Peroxide value Thiobarbituric Mold growth acid number Treatments (T) 2 0.041307** 11.579** 0.020747** 0.141448** Days (D) 2 0.301002** 77.653** 0.127100** 0.064848** T×D 4 0.009849NS 3.235NS 0.007940NS 0.009948NS Error 18 0.004837 1.116 0.003364 0.003457 Total 26 ** = Highly significant (P<0.01); Df = Degree of freedom 100 Table 4.30. Effect of treatments and storage on the water activity of sesame flour supplemented doughnuts Storage (days) Treatments S0 S3 S7 Means T0 0.691±0.37 0.712±0.43 0.720±0.52 0.707±0.38a T1 0.601±0.26 0.642±0.32 0.671±0.47 0.638±0.22b T2 0.501±0.13 0.551±0.22 0.582±0.36 0.544±0.16c Means 0.597±0.27c 0.635±0.32b 0.657±0.45a Means having same letters are statistically non-significant (P>0.05) Means ± S.D 101 The increase in water activity of products during storage is might be due to variation in humidity temperature, hygroscopic nature of food samples and packaging materials which may cause negative effect on textural characteristics and sensorical attributes (Tiwari et al., 2011). The water activity of sesame and wheat-based bread were from 0.55 to 0.72 (Jakubcyzk et al., 2008). Presence of low water activity i.e. 0.44-0.55 in extruded snacks made these products shelf stable (Katz and Labuza, 2010) whereas in composite flour breads, water activity was ranged from 0.33 to 0.56 (Akhtar et al., 2016). Food products with low water activity are considered useful especially for school nutrition programs in less developed nations having warm environmental conditions as well as meager infrastructure for the storage of commodities (Sharif et al., 2014). 4.4.6.2. Peroxide value Mean values for the peroxide value (Table 4.31) ranged from 4.84±0.60 to 10.72±0.33meq/Kg. It is apparent from the results that doughnuts containing 20g/100 of sesame flour depicted the lowest (4.84±0.60meq/Kg) peroxide value followed by doughnuts supplemented with 20g/100 of sesame flour (7.83±0.56meq/Kg) whereas the maximum value (10.72±0.33meq/Kg) was found in doughnuts without any supplementation. The lower peroxide value of doughnuts supplemented with sesame flours were might be due to presence of bioactive components i.e. sesamin, sesamol and sesamolin and phenolics. Antioxidant activities of sesame extracts were analyzed by using 2, 2-dipheny l-1-picrylhydrazyl (DPPH) methods. The white sesame varieties showed inhibiting effect against DPPH (up to 56.37%) as compared to standard commercial antioxidants i.e. BHA and TBHQ (Srinivasan, 2005). Storage also has pronounced impact on peroxide value of the stored products. In fresh samples, it was 6.73±1.14meq/kg which was gradually increased to 7.68±0.98 and 8.99±0.61meq/Kg during 3 and 7 days storage, respectively. During the storage, changes in lipid peroxidation monitored by estimating peroxide value revealed significant decrease due to the presence of antioxidants present in cereal bars (Sharma et al., 2006). In a study, marjoram powder supplemented cakes were prepared and assessed for peroxide value during the storage at room temperature. There was decrease in peroxide value of marjoram powder supplemented cakes from 12.47 to 3.40meq/Kg as compared to control. It was concluded that marjoram powder suppressed the oxidation reactions due to the presence of natural antioxidants inside the powder (Hafez, 2012). 102 Table 4.31. Effect of treatments and storage on peroxide value (meq/Kg) of sesame flour supplemented doughnuts Storage (days) Treatments S0 S3 S7 Means T0 11.70±0.83 10.82±0.71 10.63±0.38 10.72±0.33a T1 6.39±0.53 7.63±0.89 9.47±0.50 7.83±0.56b T2 3.09±0.06 4.58±0.65 6.86±0.53 4.84±0.60c Means 6.73±1.14b 7.68±0.98b 8.99±0.61a Means having same letters are statistically non-significant (P>0.05) Means ± S.D 103 4.4.6.2. Thiobarbituric acid number Means for the thiobarbituric acid number (Table 4.32) showed the maximum value in doughnuts without supplementation (0.658±0.30 mg/Kg) whereas doughnuts containing 10g/100 of sesame flour (0.499±0.22mg/Kg) and doughnuts with 20g/100g of sesame flour (0.426±0.16 mg/Kg) depicted comparatively low TBA values. Storage also has significant effect on thiobarbituric acid number of the doughnuts. At the initiation of storage, TBA value was 0.476±0.20mg/Kg which was gradually increased to 0.535±0.28 mg/Kg and 0.571±0.34 mg/Kg after 3rdand 7thday of storage. Thiobarbituric acid number is considered as a standard marker for determining the lipid peroxidation changes during storage. In an earlier study, there was increase in the TBA value (0.270 to 0.517mg/Kg) of cakes supplemented with marjoram powder during the storage. It indicates that the natural antioxidants and some bioactive components incorporated into cakes exhibited prevention of lipid oxidation during storage (Hafez, 2012). Similarly, the production of oxidation products during frying of meat products (measured using TBARS assay)can be reduced by adding antioxidants to stop lipid peroxidation during the storage (Xiong et al., 2015). 4.4.6.3. Mold count Results for the mold count (Table 4.33) exhibited maximum growth (0.463×102±0.33 CFU/g) in doughnuts without supplementation followed by doughnuts containing 10g/100 of sesame flour (0.308×102±0.27 CFU/g) whereas the minimum count was observed in doughnuts with 20g/100g of sesame flour (0.327×102±0.19 CFU/g). There was decrease in mold growth in doughnuts containing various levels of sesame flour. This was might be due to low water activity of these treatments which has inhibitory effect on growth of micro-organisms. Storage has significant effect on mold in doughnuts. At the start of study, it was (0.258×102±0.23CFU/g) which was gradually increased to 0.337×102±0.34 CFU/g and 0.503×102±0.46 CFU/g at the mid and end of study, respectively. There was increase in mold growth (0.30 to 0.66×102CFU/g) of cereals and nuts bars developed with sesame, oat flakes, skim milk powder and almonds during storage (Al-Hooti et al., 1997). Breads developed with chicken meat and their powders werecrispy in taste and studied for mold count. The results showed that aerobic plate count and yeast-mold count remained within the permissible ranges (Yah et al., 2009). Similarly, there was no mold growth on the bread during the first three days. However, mold growth appeared with the passage of time (Collins et al., 1991). 104 Table 4.32. Effect of treatments and storage on thiobarbituric acid number (mg/kg) of sesame flour supplemented doughnuts Storage (days) Treatments S0 S3 S7 Means T0 0.661±0.19 0.665±0.27 0.649±0.65 0.658±0.30a T1 0.446±0.08 0.498±0.19 0.552±0.54 0.499±0.22b T2 0.322±0.05 0.443±0.09 0.513±0.42 0.426±0.16c Means 0.476±0.20b 0.535±0.28a 0.571±0.34a Means having same letters are statistically non-significant (P>0.05) Means ± S.D 105 Table 4.33. Effect of treatments and storage on the mold count (×102 CFU/g) of sesame flour supplemented doughnuts Storage (days) Treatments S0 S3 S7 Means T0 0.280±0.15 0.460±0.31 0.650±0.55 0.463±0.33a T1 0.243±0.27 0.260±0.16 0.420±0.39 0.308±0.27b T2 0.250±0.35 0.290±0.27 0.440±0.24 0.327±0.19c Means 0.258±0.23c 0.337±0.34b 0.503±0.46a Means having same letters are statistically non-significant (P>0.05) Means ± S.D 106 4.5. Biological Evaluation of Sesame Flour Supplemented Doughnuts Sesame flour supplemented doughnuts were studied for nutritional quality through protein quality bioassay using male Sprague Dawly rats. This approach is considered useful for a uniform comparison of experimental diets containing sesame with reference diet followed by their response on growth assessing protein efficiency ratio (PER), net protein ratio (NPR), true digestibility (TD), biological value (BV) and net protein utilization (NPU). 4.5.1. Growth study parameters of experimental diets Growth study parameters i.e., PER and NPR exhibited significant variations among the experimental groups fed with test diets containing doughnuts supplemented with sesame along with reference diet (Table 4.34). Means for protein efficiency ratio (Table 4.35) showed maximum value (2.43±0.29) in rats fed on control diet i.e. soy protein whereas among the experimental diets, the highest PER (0.86±0.17) was noticed in rats fed with diet containing (20g/100g) sesame supplemented doughnuts (G2). Protein consumed by individuals in different proportions from diverse regions can result in variations in the amino acids concentrations, which ultimately alter its efficiency. Findings of this study were in conformance with the research conducted on wheat flour supplemented with soy protein. The results showed improved protein efficiency ratio (70%) in experimental group in comparison with casein group (Rodriguezet al., 2001). Similarly, in another investigation, in rats fed with 20% soy supplemented diets PER was near to 0.30 (Iwata et al., 1990). Likewise, snacks produced with single cell protein by Spirulina platensis showed PER upto 1.72 (Moreira et al., 2011). Means for net protein utilization (Table 4.35) revealed maximum value (5.14±0.38) in rats fed with diet containing soy protein followed by 2.81±0.25 in rats fed with diet having 20g/100g sesame supplemented doughnuts. It is obvious from the results that diets containing soy performed better than diets based on sesame as reflected by the growth response from Sprague Dawly rats. It is the ratio of individual protein intake of test group and the weight gain as compared to the group containing no protein diet (FAO, 2016). 107 Table 4.34. Mean squares for PER and NPR of experimental diets SOV Df PER NPR Diets 2 4.4462** 13.156** Error 12 0.0052 0.011 Total 14 ** = Highly significant (P<0.01) Df = Degree of freedom NPR: Net protein ratio PER: Protein efficiency ratio 108 Table 4.35. Effect of experimental diets on PER and NPR in Sprague Dawly rats Diets PER NPR G1 0.74±0.05c 2.02±0.12c G2 0.86±0.17b 2.81±0.25b G3 2.43±0.29a 5.14±0.38a Means having same letters are statistically non-significant (P>0.05) Means ± S.D G1= Rats fed with diet containing 10% sesame flour G2= Rats fed with diet containing 20% sesame flour G3= Rats fed with control diet (soy protein) 109 4.5.2. Nitrogen balance study parameters of experimental diets Nitrogen balance study parameters i.e., true digestibility (TD), biological value (BV) and net protein utilization (NPU) showed significant differences among the test diets containing different levels of sesame supplemented doughnuts as compared to control group (Table 4.36). It is obvious form the results (Table 4.37) that maximum digestibility (64.51±0.84%) was observed in rats fed with diet containing 20g/100g of sesame flour followed by the diet containing 10g/100g sesame flour (51.36±0.94%). However, diet containing only soy protein revealed 87.85±2.01% true digestibility. Better results for digestibility of sesame-based diets mean the lowest fecal nitrogen output and highest retention rate in rat bodies. In a study, 75.5% true protein digestibility was found in animals fed with Spirulina plantensis (Narasimha et al., 1982). Means for the effect of experimental diets on biological value of Sprague Dawley rats (Table 4.37) explicit highest biological value (69.64±1.08%) in diets containing 20g/100g sesame flour supplemented doughnuts followed diet containing 10g/100g sesame flour (51.94±0.79%). Nevertheless, rats fed with soy-based diets revealed the highest biological value (90.48±2.56%). Biological value is defined as a measure of nitrogen retained for growth or maintenance of optimum health. Spirulina fed group showed 68% biological value in Wistar male albino rats ascompared to control group (Narasimha et al., 1982). In another study, rats fed with diets containing cyanobacteria showed 73.33% biological value (Zepka et al., 2010). In sesame supplemented diets, 80 to 84% biological value has been reported (Cordesse, 2012). Means for the effect of experimental diets on net protein utilization (Table 4.37) in Sprague Dawly rats exhibited the maximum value (61.32±0.96%) in the diets containing 20g/100g sesame flour supplemented doughnuts followed by diet containing 10g/100g of sesame flour (53.48±0.81%). It was further observed that the diet containing soy showed the highest net protein utilization (80.75±1.93%). In a study, net protein utilization of soy supplemented flour was 62.21% (Zepka et al., 2010). Wheat flour supplemented with spirulina indicated 90% net protein utilization as compared to casein protein (Iwata et al., 1990). Conclusively, results for both growth and nitrogen balance study revealed that addition of sesame flour in snacks may improve the nutritional quality of these products and ultimately the consumers gain benefit from it. 110 Table 4.36. Mean squares for TD, BV and NPU of experimental diets SOV Df TD BV NPU Diets 2 1707.7** 1860.8** 985.54** Error 12 9.4 13.9 8.85 Total 14 ** = Highly significant (P<0.01) BV: Biological value NPU: Net protein utilization TD: True digestibility 111 Table 4.37. Effect of experimental diets on the TD, BV and NPU in Sprague Dawly rats Diets TD (%) BV (%) NPU (%) G1 51.36±0.94c 51.94±0.79c 53.48±0.81c G2 64.51±0.84b 69.64±1.08b 61.32±0.96b G3 87.85±2.01a 90.48±2.56a 80.75±1.93a Means having same letters are statistically non-significant (P>0.05) Means ± S.D 112 4.6. Efficacy Studies in School Aged Children 4.6.1. Socio-demographic characteristics Before the conduct of efficacy studies, different questions were asked from the study participants (n=45) regarding social and demographic characteristics. About 13.3% mothers were belonging to working class whereas 86.7% were housewives. Children mental and cognitive growth is indispensable and to accomplish this task mother’s education is important. About 44.6% mothers were uneducated or having primary education, 48.8% have education of high school and only 6.6% were having graduation degree. Family size has a great impact on regular expenditures and distribution of money for food items, so respondents were asked about this too. It was observed that mostly had 3-4 children (44.4%) followed by ≤ 2 (31.2%) and remaining had ˃4 children (24.4%). Currently, media has become a very important passage of information particularly for household ladies. Nevertheless, only (55.5%) of the mothers were having awareness about healthy life and nutrition. It is assumed that parents are being considered as a manager for selections of food for their children. Children have damaging effect on their health if they are forced again and again to eat the same food that they really do not like. It was asked that who makes decision for child meal. The results recorded that majority of the participants (75.6%) answered it is the entire decision of child what they want to eat on daily basis. Healthy life persists due to the contribution of good source of drinking water and nutrition and health of child is dependent over it; filtered water intake was only 17.8%, whereas canal or underground water intake was 82.2% among the respondents. Some questions related to income resources were asked to have knowledge about the financial situations of the families. About 84.4% participants were living in their own houses whereas 15.6% were residing on rent. Cycle or motor cycle was more famous (82%) vehicle while only 6.6% have car and 11.4% even do not had any kind of vehicle. Regarding financial restraints, which have direct connection with the education and food availability, only 40% respondents have sufficient financial resources for livelihood. 4.6.2. Dietary intake assessment 24-h recall method was used for the assessment of dietary intake. Regular energy and nutrient intake of children/day was evaluated by inquiring their last 24-h food intake with the use of USDA nutrient database (Table 4.38). 113 Table 4.38. Mean daily dietary intake of study groups (n=45) through 24-h recall method Study groups Variables Energy (Kcal) Protein (g) Carbohydrate (g) Total Sugar (g) Fat (g) G1 (n=15) 1735.29±19.03 27.42±0.18 207.59±1.11 76.52±0.80 52.78±0.61 G2(n=15) 1728.22±20.87 32.54±0.24 192.67±2.01 74.16±0.55 54.43±0.56 G3(n=15) 1638.32±14.94 34.53±0.36 185.32±1.27 72.68±0.67 56.64±0.37 USDA National Nutrient Database was used as standard for data analysis 114 Results reveal that average energy intake was ranged from 1638.32 to 1735.29 Kcal, protein 27.42 to 34.53 g, carbohydrates 185.32 to 207.59 g, total sugar 72.68 to 76.52 g, and fat 52.78 to 56.64 g per subject/day. The differences in these nutrients intake was might be due to different socio- demographic characteristics among the study groups. 4.6.3. Anthropometric assessment The study volunteers were evaluated for the anthropometric measurements. Mean squares showed significant effect of protein supplemented doughnuts and study intervals on weight and body mass index but non-significant impact on height, HC and MUAC. Likewise, interactions between experimental diets and study intervals were significant for weight and BMI and non-significant for rest of the parameters (Table 4.39). 4.6.3.1. Height Means for height (Table 4.40) of school aged children exhibited the values ranged from 143.86±0.76 cm to 144.84±0.91 cm among all groups. The height of the children served with the doughnuts without supplementation increased from 143.63±1.35 cm to 145.75±2.97 cm for 60 days intervention. Similarly, the other groups served with sesame-based doughnuts presented the height ranged from 143.48±1.28 cm to 144.11±1.43 cm and 144.45±1.14 cm to 145.09±1.47cm. Overall, height was ranged from 143.85±0.71cm to 144.98±1.01 cm during the study interval. Another research was carried out to assess height of Pakistani school going children in order to develop report with varied social, financial and traditional credentials. It was cross-sectional study in a national project of Higher Education Commission (HEC), Pakistan. The children (12,837) with highest immunity, without having any prolonged infection, normal birth weight of age 3- 16years were included. The mean height of children was 139.10±18.43cm. Aziz et al. (2012) reported that at the age of 9, 10, 11 and 12 years, the height observed was, 128.01±5.4, 139.10±9.1, 141.11±8.1, 147.21±9.7cm, respectively. According to WHO standards, it is stated that the average height of the children is 136.1, 137.7, 143.1 and 149.1cm at the age of 9, 10, 11 and 12 years, respectively (WHO, 2009). 115 Table 4.39. Mean squares for the anthropometric measurements of school aged children SOV Df Height Weight BMI HC MUAC Intervals (D) 2 14.37NS 8.5579** 1.0278** 0.028NS 0.0215NS Diets (T) 2 11.45NS 6.1267** 0.9828** 1.327NS 0.4515NS D x T 4 3.43NS 0.6458* 0.4812* 0.003NS 0.0065NS Error 126 25.81 0.2290 0.1695 3.689 0.4399 Total 134 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01) BMI=Body mass index Df = Degree of freedom HC=Head circumference MUAC=Mid upper arm circumference 116 Table 4.40. Effect of protein enriched sesame fortified doughnuts on height of school aged children Study intervals Height (cm) Means (Days) G1 G2 G3 S0 143.63±1.35 143.48±1.28 144.45±1.14 143.85±0.71 S30 144.25±1.82 143.98±1.39 144.98±1.19 144.40±0.89 S60 145.75±2.97 144.11±1.43 145.09±1.47 144.98±1.01 Means 144.54±0.69 143.86±0.76 144.84±0.91 Means± S.D S0= 0 day of study; S30= 30 days of study; S60= 60 days of study G1= Children provided with doughnuts without supplementation G2= Children provided with doughnuts containing 10% sesame flour G3= Children provided with doughnuts containing 20% sesame flour 117 4.6.3.2. Weight Mean results for weight (Table 4.41) of the school aged children exhibited the values ranged from 37.58±0.07 to 38.30±0.23 Kg among the study groups. Children served with doughnuts without supplementation revealed weight ranged from 37.34±0.12 to 37.84±0.23 Kg for 60 days study. Likewise, children consuming 10g/100g of sesame flour supplemented doughnuts have 37.42±0.09 Kg weight at the start of study which was increased to 38.73±0.22 Kg at the end of the study. Children served with doughnuts containing 20g/100g showed maximum increase in weight (1 kg) followed by doughnuts with 10g/100g of sesame flour. Overall, weight was varied from 37.57±0.09kg to 38.44±0.21 Kg during the study. It was explained in a study reported by Aziz et al., 2012 that the average weight of Pakistani children aged 9-12 years ranged from 26.10±0.41 Kg to 38.11±8.7Kg. Similarly, World Health Organization standards indicated that the average weight of the children of age 9-12 years should be 30.30 to 41.38Kg (WHO, 2009). In a similar research, fortified spreads were served to Malawian infants for 12 weeks. The results indicated substantial increase in their body weight (1.5Kg) due to the consumption of protein enriched diet (Kuusipalo et al., 2006). In another study, the weight of school going children served with extruded snacks containing more dietary fiber, protein and micronutrients, was increased from 37.53 to 38.23 Kg after 60 days (Shah, 2016). 4.6.3.3. Body mass index (BMI) BMI (Table 4.42) values of the school children were ranged from 18.03±0.35 to 18.32±0.43 Kg/m2 among the study groups. Means for BMI of children served with doughnuts without supplementation were increased from 17.68±0.07 to 18.27±0.71 Kg/m2 after 60 days. Similarly, children served with doughnuts containing 10g/100g of sesame flour revealed 18.17±0.52 Kg/m2 BMI at the initiation of the trial which was subsequently increased to 18.23±0.27 Kg/m2 at the end of the study. BMI of the children served with 20g/100g of sesame flour supplemented doughnuts increased to 18.54±0.26kg/m2 during the intervention period. It is apparent from the results that during the intervention period, maximum increase (0.55kg/m2) was depicted in the children served with doughnuts containing 10g/100g of sesame flour followed by the children served with doughnuts developed with 20g/100g of sesame flour (0.29kg/m2). Overall, BMI was varied from 18.03±0.17 to 18.34±0.38kg during the whole study. 118 Table 4.41. Effect of protein enriched sesame fortified doughnuts on weight of school aged children Study intervals Weight (Kg) Means (Days) G1 G2 G3 S0 37.34±0.12e 37.42±0.09e 37.95±0.18bc 37.57±0.09c S30 37.56±0.16de 38.03±0.12bc 38.21±0.21b 37.93±0.17b S60 37.84±0.23cd 38.73±0.22a 38.75±0.37a 38.44±0.21a Means 37.58±0.07c 38.06±0.10b 38.30±0.23a Means having same letters are statistically non-significant (P>0.05) Means± S.D 119 Table 4.42. Effect of protein enriched sesame fortified doughnuts on body mass index (BMI) of school aged children Study intervals BMI (Kg/m2) (Days) Means G1 G2 G3 S0 17.68±0.07c 18.17±0.52b 18.25±0.12ab 18.03±0.17c S30 18.18±0.12b 18.23±0.63ab 18.17±0.18b 18.19±0.26b S60 18.23±0.27b 18.27±0.71b 18.54±0.26a 18.34±0.38a Means 18.03±0.35b 18.22±0.26a 18.32±0.43a Means having same letters are statistically non-significant (P>0.05) Means± S.D 120 WHO Standards reveal that children at the age of 9-12 years have average BMI (16.1-17.5kg/m2) in the range from (WHO, 2009) whereas average BMI of Pakistani children of the same age is 16.75±3.21kg/m2 (Aziz et al., 2012). In another research, children were assessed on the basis of their weight and height after serving them with fortified complementary foods and 0.12-0.25% increase in the BMI was revealed (Sirkka et al., 2018). 4.6.3.4. Head and mid upper arm circumference The assessment of the head circumference in experimental groups revealed non-significant differences during the study interval. Means for the head circumference (Table 4.43) were ranged from 52.27±0.45cm to 52.61±0.53 cm in all study groups. Overall, head circumference was ranged from 52.41±0.27cm to 52.46±0.42cm during the 60 days intervention. The results of the present research are related to previous findings, which revealed relationship between head circumference and brain volume in toddlers, children and normal adults (Bartholomeusz et al., 2002). The results of this study showed that head circumference of the children aged 9-12 years were ranged from 52.31±0.04 to 54.12±0.14cm. Means for MUAC (Table 4.44) of children revealed values ranged from 19.42±0.23 to 19.62±0.35 cm. During 60 days of study, means for MUAC were ranged from 19.50±0.11 to 19.55±0.31 cm. In a cross-sectional study, Turkish children and adolescents were assessed to determine standard values of MUAC. Students (5,553) aged 6-17 years were chosen by a random sampling from different schools of city center, urban and rural areas of Kayseri, Central Anatolia. The mid upper arm circumference 50th percentile was ranged from 17.6 to 23.6cm in males and 15.6 to 20.9cm in girls (Ozturk et al., 2009). Results of another investigation conducted in Southern Iran for establishing reference values of MUAC were 18.51±2.3cm in school going children (Ayatollahi, 2012). 4.6.4. Blood Analysis Mean squares for the effect of sesame flour supplemented and fortified doughnuts (Table 4.45) revealed significant differences among the experimental groups with respect to serum ferritin and serum zinc levels in school aged children. Similarly, study intervals and interaction between study intervals and diets also showed significant differences. 121 Table 4.43. Effect of protein enriched sesame fortified doughnuts on head circumference(HC) of school aged children Study intervals HC (cm) Means (Days) G1 G2 G3 S0 52.23±0.40 52.42±0.38 52.59±0.44 52.41±0.27 S30 52.27±0.64 52.44±0.47 52.62±0.52 52.44±0.30 S60 52.31±0.73 52.45±0.54 52.63±0.65 52.46±0.42 Means 52.27±0.45 52.44±0.36 52.61±0.53 Means± S.D 122 Table 4.44. Effect of protein enriched sesame fortified doughnuts on mid upper arm circumference (MUAC) of school aged children Study intervals MUAC (cm) Means (Days) G1 G2 G3 S0 19.41±0.12 19.49±0.20 19.61±0.15 19.50±0.11 S30 19.42±0.22 19.52±0.34 19.62±0.22 19.52±0.22 S60 19.43±0.31 19.58±0.42 19.63±0.34 19.55±0.31 Means 19.42±0.23 19.53±0.10 19.62±0.35 Means±S.D 123 Table 4.45. Mean squares for the serum ferritin and serum zinc levels of school aged children SOV Df Serum Ferritin Serum Zinc Intervals (D) 2 366.51** 13.2527** Diets (T) 2 67.16** 1.9807** D x T 4 05.33** 1.3019** Error 126 1.25 0.1203 Total 134 ** = Highly significant (P<0.01); Df = Degree of freedom 124 4.6.4.1. Serum ferritin Means values for serum ferritin levels in children (Table 4.46) exhibited maximum ferritin concentration in the group served with 20g/100g of sesame flour supplemented doughnuts (32.71±0.45µg/L) followed by 10g/100g of doughnuts (31.50±0.40µg/L). However, minimum value (30.26±0.31µg/L) for serum ferritin was observed in doughnuts without supplementation. The variation in serum ferritin levels were might be due to the differences in iron content of raw materials used for supplementation. At the start of study, serum ferritin was 28.60±0.15µg/L which was increased to 34.31±0.36µg/L at the end of the study. Percent change is shown in Figure 4.2 among different groups. There was 7.4 to13.3% change observed among all groups for 60 days study due to iron fortification and supplementation of sesame flour. It was increased from 8.6 to 13.3% in children served with doughnuts having 20g/100g of sesame flours followed by 9.5 to 10.2% in children consuming 10g/100g of sesame flour doughnuts. The maximum improvement was observed in doughnuts supplemented with 20g/100g of sesame flour due to the higher levels of iron content in raw materials. These results were in conformance with the maximum level of hemoglobin in the same group (Table 4.49). The serum ferritin is the amount of stored iron in body. Its Deficiency causes anemia due to which body does not have sufficient amount of red blood cells and not able to even work properly. Findings of another study conducted on primary school children (N=115) of age 6-11 years served with iron fortified biscuits for 11 months revealed decrease in serum ferritin concentration from 27.7 to 13.8% (Goyle and Prakash, 2010). Likewise, Sri Lankan children (age 6-10 years) were assessed for the impact of zinc and iron fortified rice. The results indicated increase (46.7 to 55.5µg/L)in serum ferritin levels (Hettiarachchi et al., 2004). 4.6.4.2. Serum zinc Results for serum zinc (Table 4.47) showed highest value (10.11±0.22 µmol/L) in group served with doughnuts developed with 20g/100g sesame flour followed by doughnuts having 10g/100g sesame flour (9.75±0.10µmol/L). The variation in serum zinc levels between the groups were might be due to the different levels of supplemented flours in the doughnuts. Initially, the mean values for serum zinc were 9.42±0.08 µmol/L, which were increased to 9.72±0.15 and 10.47±0.34µmol/L after 30 and 60 days study interval, respectively. 125 Table 4.46. Effect of protein enriched sesame fortified doughnuts on serum ferritin of school aged children Study intervals Serum ferritin (µg/L) Means (Days) G1 G2 G3 S0 28.07±0.18g 28.56±0.15fg 29.18±0.25f 28.60±0.15c S30 30.14±0.24e 31.47±0.27d 33.05±0.34c 31.55±0.24b S60 32.58±0.33c 34.46±0.36b 35.89±0.45a 34.31±0.36a Means 30.26±0.31c 31.50±0.40b 32.71±0.45a Means having same letters are statistically non-significant (P>0.05) Means± S.D 126 14 Seum ferritin 13.3 12 10.2 10 9.5 8.6 8.1 8 7.4 30 Days 6 60 Days % Change% 4 2 0 G1 G2 G3 Study groups Figure 4.2. Percent increase in serum ferritin of school aged children 127 Table 4.47. Effect of protein enriched sesame fortified doughnuts on serum zinc of school aged children Study intervals Serum zinc (µmol/L) Means (Days) G1 G2 G3 S0 9.16±0.09d 9.05±0.09d 10.05±0.10b 9.42±0.08c S30 9.69±0.18c 9.71±0.11c 9.76±0.21c 9.72±0.15b S60 10.41±0.29a 10.48±0.23a 10.53±0.32a 10.47±0.34a Means 9.74±0.09b 9.75±0.10b 10.11±0.22a Means having same letters are statistically non-significant (P>0.05) Means± S.D 128 9 Serum zinc 8 7.9 7 6.9 6.4 6 5.9 4.9 5 4.8 4 30 Days % Change% 60 Days 3 2 1 0 G1 G2 G3 Study groups Figure 4.3. Percent increase in serum zinc of school aged children 129 Percent change in serum zinc is shown in Figure 4.3 among different groups. There was 4.8-7.9% increase in serum zinc due to fortification. It was increased in children served with 20g/100g 0f sesame flour from 4.9 to 7.9% at 60 days followed by children served with 10g/100g of sesame flour 6.4 to 6.9% at 30 days. Maximum improvement was noted in doughnuts supplemented with 20g/100g of sesame, it was due to the maximum zinc content in the raw materials. Zinc serves as a cofactor for different enzymes i.e. alkaline phosphatase, alcohol dehydrogenase, polymerases, carbonic anhydrase and many other physiologically important proteins. Different enzymes are more sensitive towards zinc depletion i.e. kinases, peptidases and phosphorylases etc. It is a vital element for wound healing as well. Its depletion occurs due to the interference of excess iron or copper with it, they cause less absorption of zinc from the diet. Its deficiency occurs in diet just because of its absence as it is only bound to phytate and provides fiber so not available for absorption through diet in the body. It is used a source of dietary supplement and found in leguminous foods (Bhowmik, et al., 2010). In a research conducted on children of Indonesia who were served with fortified wheat flour containing meal and it was found that there was 24% zinc absorption occurred in them from this diet (Herman et al., 2002). In another study carried out in children of Sri Lanka of age 6-11 years in order to check the enhanced effect of zinc and iron content in fortified rice. Results indicated that serum zinc was ranged from 12.1-13.1µmol/L (Hettiarachi et al., 2004). 4.6.4.3. Hemoglobin (Hb) Means squares (Table 4.48) for the effect of sesame supplemented and micro-nutrient fortified doughnuts on complete blood count (CBC) of school children revealed significant differences among the study intervals regarding all CBC parameters except for WBC, neutrophils, lymphocytes and monocytes. Similarly, treatments also showed significant differences regarding HB, RBC, PCV, MCV, MCH, MCHC and WBC except for platelets. The interaction between study intervals and experimental diets exhibited non-significant results in all parameters. Means for effect of sesame-based doughnuts on Hb of children (Table 4.49) depicted maximum value (13.54±0.48 g/dL) in group served with 20g/100g of sesame flour-based doughnuts followed by 10g/100g of sesame flour doughnuts (13.25±0.34 g/dL). At the baseline of study, mean Hb was (12.75±0.16 g/dL) and it was enhanced to 13.96±0.35 g/dL at the end of trial. % change in Hb of children eating supplemented doughnuts (Figure 4.4) indicated maximum increase (6.3%) in the group served with 20g/100g of sesame-based doughnuts followed by 10g/100 of sesame doughnuts 130 (4.7%) at the end of intervention. The results are in conformance with the findings of serum ferritin. The maximum increase in Hb of children served with the addition of sesame flour was due to the presence of higher level of protein and iron concentrations. This test is used to detect, observe and screen diverse ailments that affect the RBC. A 70 days trial in infants (N=56) served with fortified products indicated improvement in Hb from 10.06 to 12.8g/dL (Saarinen et al., 2008). However, Hb of school aged Sri Lankan was ranged from 12.2-13.1g/dL during the study (Hettiarachchi et al., 2004). In another study, 6-11 years old primary school children (N=115) served with iron fortified biscuits exhibited significant improvements in Hb levels after 40 months (Stuijvenberg et al., 2001). 4.6.4.4. Red blood cell (RBC) Effect of sesame fortified doughnuts on RBC (Table 4.50) revealed values ranged from 4.56±0.23 to 4.67±0.44 Mill/Cu.mm among all the experimental groups. Maximum value (4.67±0.44Mill/Cu.mm) was found in the group served with 20g/100g of sesame-based doughnuts. At the start of study, mean value was (4.45±0.13Mill/Cu.mm) which was increased to 4.78±0.37 Mill/Cu.mm after 60 days trial. Percent increase in RBC is depicted in Figure 4.5. Maximum increase was observed in the children served with 10g/100g of sesame flour at 30 day (7.8%) whilst at the end of study, maximum increase (4.1%) was observed in children served with 20g/100g of sesame flour doughnuts. It is a biomarker in folic acid, Vitamin B12 and iron fortification programs in which an increase or decrease must be explained with other parameters i.e. hematocrit, red cell indices hemoglobin, and reticulocyte count. A study was conducted on 56 infants consuming iron supplemented diets. The results explicit 4.7×1012µmol/L RBC at the end of dietary intervention (Saarinen et al., 2008). Findings of another study revealed 0.391-4.88µmol/L RBCs in healthy men of age 24-57 years served with vitamin fortified diets (Nair and Krishnaswamy, 2001). 131 Table 4.48. Mean squares for complete blood count (CBC) of school aged children SOV Df Hb RBC PCV MCV MCH MCHC Intervals (D) 2 16.5727** 1.2345** 162.754** 719.84** 51.741** 73.112** Diets (T) 2 1.2912* 0.1505* 36.456** 1079.99** 67.203** 1.173NS D x T 4 0.6397NS 0.0980NS 4.413NS 83.57NS 3.816NS 2.926NS Error 126 0.2836 0.0422 1.956 42.50 1.838 2.057 Total 134 SOV Df Platelets WBC Neutrophils Lymphocytes Monocytes Eosinophils Intervals D) 2 3085850000** 868446NS 272.09NS 28.66NS 0.4155NS 7.0245** Diets (T) 2 316550000NS 770707** 514.38** 32.13* 7.2780** 1.4415** D x T 4 100900000NS 202636NS 10.06NS 0.94NS 0.0398NS 0.1898NS Error 126 110804855 313860 92.74 10.05 0.1413 0.0854 Total 134 Df = Degree of freedom ** = Highly significant (P<0.01) NS = Non-significant (P>0.05) * = Significant (P<0.05) Hb= Hemoglobin MCHC= Mean corpuscular hemoglobin concentration MCV= Mean cell volume or mean corpuscular volume PCV= Packed cell volume RBC= Red blood cell WBC= White blood cells 132 Table 4.49. Effect of protein enriched sesame fortified doughnuts on hemoglobin of school aged children Study intervals Hb (g/dL) Means (Days) G1 G2 G3 S0 12.87±0.10 12.46±0.14 12.91±0.17 12.75±0.16c S30 13.23±0.13 13.32±0.21 13.43±0.32 13.33±0.29b S60 13.65±0.18 13.95±0.32 14.28±0.41 13.96±0.35a Means 13.24±0.19b 13.25±0.34b 13.54±0.48a Means having same letters are statistically non-significant (P>0.05) Means± S.D 133 8 Hb 7 6.9 6.3 6 5 4.7 4.0 4 30 Days 3.2 % Change % 3 2.8 60 Days 2 1 0 G1 G2 G3 Study groups Figure 4.4. Percent increase in Hb of school aged children 134 Table 4.50. Effect of protein enriched sesame fortified doughnuts on red blood cells (RBCs) of school aged children Study intervals RBC (Mill/Cu.mm) Means (Days) G1 G2 G3 S0 4.48±0.04 4.38±0.14 4.49±0.09 4.45±0.13c S30 4.53±0.15 4.72±0.27 4.67±0.18 4.64±0.24b S60 4.67±0.25 4.81±0.34 4.86±0.32 4.78±0.37a Means 4.56±0.23b 4.64±0.36ab 4.67±0.44a Means having same letters are statistically non-significant (P>0.05) Means± S.D 135 9 RBC 8 7.8 7 6 5 4.1 4.0 4 30 Days % Change% 3.1 3 60 Days 2 1.9 1.1 1 0 G1 G2 G3 Study groups Figure 4.5. Percent increase in RBC of school aged children 136 4.6.4.5. Packed cell volume (PCV) Means for packed cell volume (Table 4.51) were ranged from 35.59±0.24 to 37.27±0.48% among the experimental groups. Maximum value (37.27±0.48%) was obtained in group of children served with 20g/100 of sesame flour doughnuts. For 60 days study, PCV was increased from 34.33±0.18 to 38.13±0.37%. Changes in PCV percentage during the study are shown in Figure 4.6. Maximum increase (6.7-6.9%) within the groups was observed in children served with doughnuts 20g/100g of sesame flour followed by the children consuming 10g/100g of sesame flour (5.0-5.8%) while minimum increase was noticed in the group eating doughnuts without supplementation (3.7-4.2%) during the study intervals. The increase in PCV was attributed to the presence of more protein and iron contents in the raw materials. This test is used to count the number of cells present in the blood. A study was conducted in infants to check the developmental changes in cells by serving iron supplemented diets. The outcomes of the intervention revealed 37.01±0.31% PCV (Saarinen et al., 2008). Another research was carried out in school aged children who were served with iron fortified biscuits and their nutritional status was checked before and after eating fortified samples for 10 months. The results indicated that PCV was increased from 35.5 to 36.4% at the end of study (Stuijvenberg et al., 2001). 4.6.4.6. Mean corpuscular volume (MCV) Results for effect of sesame enriched fortified doughnuts on MCV of school aged children (Table 4.52) showed the values ranged from 75.33±0.66 fL to 84.81±1.38 fL among all groups. The highest MCV (84.81±1.38 fL) was found in the group fed with 20g/100 of sesame flour supplemented doughnuts Furthermore, mean values for MCV were 76.47±0.74 fL at the initiation of study which were increased to 84.37±1.42 fL at the end of the study. Percent change in different groups at the mid and end of 60 days trial are illustrated in Figure 4.7. Children served with 10- 20g/100g defatted sesame flour indicated 5.1-6.4% improvement in the MCV whereas, the minimum increase was noted in the group consuming only fortified doughnuts without supplementation (1.4-4.6%). MCV is the mean value of red cells in blood. It fluctuated according to the amount of red blood cells. Small, normal and large size RBC indicates microcytic, normocytic and macrocytic MCV. 137 Table 4.51. Effect of protein enriched sesame fortified doughnuts on packed cell volume (PCV) of school aged children Study intervals PCV (%) Means (Days) G1 G2 G3 S0 34.19±0.14 33.96±0.24 34.83±0.30 34.33±0.18c S30 35.63±0.31 35.93±0.35 37.23±0.50 36.26±0.25b S60 36.94±0.42 37.71±0.43 39.74±0.36 38.13±0.37a Means 35.59±0.24b 35.87±0.31b 37.27±0.48a Means having same letters are statistically non-significant (P>0.05) Means± S.D 138 8 PCV 6.9 7 6.7 6 5.8 5 5.0 4.2 4 3.7 30 Days % Change% 3 60 Days 2 1 0 G1 G2 G3 Study groups Figure 4.6. Percent increase in PCV of school aged children 139 Table 4.52. Effect of protein enriched sesame fortified doughnuts on mean corpuscular volume (MCV) of school aged children Study intervals MCV (fL) Means (Days) G1 G2 G3 S0 73.18±0.70 76.71±0.67 79.53±1.68 76.47±0.74c S30 76.93±1.28 82.91±1.96 84.74±2.29 81.53±1.18b S60 75.89±1.19 87.06±2.59 90.16±2.67 84.37±1.42a Means 75.33±0.66b 82.23±1.26a 84.81±1.38a Means having same letters are statistically non-significant (P>0.05) Means± S.D 140 9 MCV 8.1 8 7 6.6 6.4 6 5.1 5 4.6 30 Days 4 % Change% 60 Days 3 2 1.4 1 0 G1 G2 G3 Study groups Figure 4.7. Percent increase in MCV of school aged children 141 Results are in conformance with the research conducted in infants (N=56) fed with iron supplemented diet to check the status of developmental changes on MCV during the trial. Results of this study showed that the average MCV was 77.8±0.4fL (Saarinen et al., 2008). 4.6.4.7. Mean corpuscular hemoglobin Means for the effect of experimental diets on MCH of children (Table 4.53) revealed the highest value (30.51±0.47Pg) in the group of children served with doughnuts developed with 20g/100g of sesame flour followed by the group consuming doughnuts containing 10g/100g of sesame flour (29.46±0.35Pg). For 60 days study, the mean corpuscular hemoglobin was ranged from 28.22±0.10 to 30.35±0.32Pg. Percent improvement during the intervention is shown in Figure 4.8. The maximum increase was observed in group served with 20g/100 of sesame-based doughnuts (4.6-5.1%) followed by 10g/100g of sesame flour supplemented doughnuts (4.1-4.5%). MCH has strong correlation with hemoglobin. Raw materials contain higher level of iron contents and they cause higher levels of MCH in sesame-based groups. MCH is defined as the mean value of hemoglobin inside a RBC. Small sized red cells would have a lower value of MCH. In a study, MCH of the children fed with iron supplemented diet showed the value of MCH in the range of 26.7±0.3Pg (Saarinen et al., 2008). 4.6.4.8. Mean corpuscular hemoglobin concentration (MCHC) Means for the effect of protein enriched and micronutrient fortified sesame-based doughnuts (Table 4.54) exhibited the maximum value (38.68±0.49%) in the group of children served with doughnuts having 20g/100 of sesame flour followed by 10g/100 of sesame (38.47±0.36%). Additionally, mean value for MCHC was 37.11±0.19% at the initiation of the study which was increased to 39.60±0.34% at the end of 60 days intervention. Percent changes (Figure 4.9) in the groups during the study were 0.5 to 5.4. Within the groups, the maximum increase (3.2-5.4%) was in the children served with 20g/100g of sesame followed by 2.4-4.9% in kids consuming product containing 10g/100 of defatted sesame flour. Minimum improvement (0.5-3.5%) was found in the group eating doughnuts having zero supplementation of sesame flour. In a study, developmental changes in red blood cells count of children were examined after provision of iron fortified biscuits. The results indicated MCHC value in the range of 34.31±1.4% (Saarinen et al., 2008). 142 Table 4.53. Effect of protein enriched sesame fortified doughnuts on mean corpuscular hemoglobin (MCH) of school aged children Study intervals MCH (Pg) (Days) Means G1 G2 G3 S0 27.38±0.30 28.21±0.24 29.06±0.29 28.22±0.10c S30 28.41±0.39 29.47±0.39 30.54±0.49 29.47±0.24b S60 28.43±0.48 30.69±0.46 31.93±0.53 30.35±0.32a Means 28.07±0.20c 29.46±0.35b 30.51±0.47a Means having same letters are statistically non-significant (P>0.05) Means± S.D 143 6 MCH 5.1 5 4.6 4.5 4.1 4 3.8 3 30 Days % Change% 2 60 Days 1.2 1 0 G1 G2 G3 Study groups Figure 4.8. Percent increase in MCH of school aged children 144 Table 4.54. Effect of protein enriched sesame fortified doughnuts on mean corpuscular hemoglobin concentration (MCHC) of school aged children Study intervals MCHC (%) (Days) Means G1 G2 G3 S0 37.42±0.36 36.95±0.32 36.95±0.29 37.11±0.19c S30 38.74±0.42 38.77±0.48 38.93±0.44 38.81±0.23b S60 38.94±0.50 39.69±0.52 40.17±0.56 39.60±0.34a Means 38.37±0.24c 38.47±0.36b 38.68±0.49a Means having same letters are statistically non-significant (P>0.05) Means± S.D 145 6 MCHC 5.4 5 4.9 4 3.5 3.2 3 2.4 30 Days % Change% 2 60 Days 1 0.5 0 G1 G2 G3 Study groups Figure 4.9. Percent increase in MCHC of school aged children 146 4.6.4.9. Platelet count and white blood cell (WBC) Results for the effect of sesame fortified doughnuts on platelet count (Table 4.55) revealed results ranged from 356800±1596 to 362033±2006numbers/Cu.mm. During the study trial, there was overall increase in platelets of all experimental groups. The maximum increase (362033±2006numbers/Cu.mm) was observed in children served with 20g/100g of sesame doughnuts followed by 10g/100g of sesame flour. Whereas, the minimum value (356800±1596numbers/Cu.mm) was found in groups served doughnuts without supplementation. At the initiation of study, the mean value was (349700±1691numbers/Cu.mm) which was increased to 365067±1734numbers/Cu.mm after 60 days of intervention. There was increase in platelets of group fed with 20g/100g of sesame flour due to the boosted nutrients in the raw materials. The normal platelet count is in the range from 1,50,000 to 4,000,00 numbers/Cu.mm (Nair and Krishnaswamy, 2001). Means for the effect of protein enriched and sesame fortified doughnuts depicted WBC ranged from 8866.1±69.7to 9656.8±114.3numbers/Cu.mm (Table 4.55). Similar findings of a research conducted on children fed with iron fortified biscuits to check the developmental changes on WBC revealed the mean values 8.3×109 to 6.8×109 in test group which were reduced to the ranges 7.5×109 to 7.4×109. WBC has normal values in the ranges of 4000-11000/Cu.mm (Stuijvenberg et al., 2001). 4.6.4.10. Neutrophils and lymphocytes Means for the effect of protein enriched and fortified sesame doughnuts expressed neutrophils levels in the children ranged from 54.27±1.59 to 60.65±2.59% (Table 4.56). The highest value (60.65±2.59%) was observed in the group nourished with doughnuts containing 20g/100g of sesame flour followed by 10g/100g of sesame-based doughnuts (55.52±1.49%). However, the neutrophil levels were the lowest (54.27±1.20%) in the group served doughnuts without supplementation of sesame. In a study, the standard values of neutrophils were 51-70% (Haddad et al., 1999). Results for the effect of sesame supplemented doughnuts on lymphocytes demonstrated mean values ranged from 36.62±0.39 to 38.28±0.59% (Table 4.56). Results showed the maximum value (38.28±0.59%) in children fed with doughnuts without supplementation followed by 10g/100g of sesame (37.70±0.41%) and 20g/100g sesame flour (36.62±0.39%). 147 Table 4.55. Effect of protein enriched sesame fortified doughnuts on plateletsand white blood cells (WBCs) of school aged children Study intervals Platelets (numbers/Cu.mm) Means (Days) G1 G2 G3 S0 350300±2725 347200±2775 351600±3330 349700±1691c S30 360200±2516 363100±2036 364900±2201 362733±1308b S60 359900±2395 365700±3109 369600±3089 365067±1734a Means 356800±1596 358667±1950 362033±2006 Study intervals WBC (numbers/Cu.mm) Means (Days) G1 G2 G3 S0 8707.3±155.7 9193.8±87.10 9396.1±82.20 9099.1±77.40 S30 8881.1±85.20 9216.5±92.90 9684.3±88.50 9260.6±70.60 S60 9009.9±105.2 9226.8±123.3 9890.1±315.8 9375.6±128.7 Means 8866.1±69.7c 9212.4±57.8b 9656.8±114.3a Means having same letters are statistically non-significant (P>0.05) Means± S.D 148 Table 4.56. Effect of protein enriched sesame fortified doughnuts on neutrophils and lymphocytes of school aged children Study intervals Neutrophils (%) Means (Days) G1 G2 G3 S0 52.39±0.97 53.49±1.06 57.29±3.15 54.39±1.17 S30 54.21±4.22 55.47±3.20 60.58±2.57 56.75±1.96 S60 56.22±2.15 57.61±1.24 64.09±1.68 59.31±2.11 Means 54.27±1.20b 55.52±1.49b 60.65±2.59a Study intervals Lymphocytes (%) Means (Days) G1 G2 G3 S0 38.89±1.47 38.84±0.79 37.34±0.58 38.36±0.59 S30 38.27±0.94 37.55±0.57 36.62±0.68 37.48±0.43 S60 37.69±0.45 36.71±0.69 35.89±0.75 36.76±0.38 Means 38.28±0.59a 37.70±0.41ab 36.62±0.39b Means having same letters are statistically non-significant (P>0.05) Means± S.D 149 During the study interval, mean values for lymphocytes varied non-significantly and were ranged from 38.36±0.59 to 36.76±0.38%. In a study, the lymphocytes were ranged from 21 to 50%(Haddad et al., 1999). 4.6.4.11. Monocytes and eosinophils Mean results for monocytes and eosinophil (Table 4.57) in the groups served with sesame-based protein enriched doughnuts depicted values ranged from 2.14±0.25 to 2.92±0.37% and 2.51± 0.34 to 2.86±0.47%, respectively. Likewise, for 60 days study trial, means for monocytes and eosinophils in the children varied insignificantly from 2.50±0.09 to 2.69±0.26% and 2.27±0.12 to 3.06±0.37, respectively. In another study, the reference range for eosinophil was 0 to 6.2% in experimental groups served with fortified diets (Haddad et al., 1999). 4.6.4.12. Serum Electrolytes Means squares for the effect of protein enriched and sesame fortified doughnuts on serum electrolytes (Table 4.58) exhibited non-significant variations among the experimental groups regarding blood chloride and potassium. Likewise, there were significant differences among the groups for blood potassium during 60 days study. However, the interactions between study intervals and experimental diets were non-significant. Mean values for serum sodium (Table 4.59) were ranged from 138.21±0.62 to 140.68±0.83mmol/L. At the start of trial, it was 138.21±0.62 mmol/L, which was increased to 140.68±0.83mmol/L at the end of the study. In a study, blood sodium was tested in different healthy individuals and results indicated that the normal values for blood sodium are 136-145mmol/L (Pramina et al., 2013). Means for the blood chloride (Table 4.59) in school aged children were 105.04±0.56 to 105.40±0.83mmol/L. Pramina et al. (2013) has reported normal ranges of blood chloride (92-112mmol/L) in healthy individuals. Mean values for blood potassium in school children were ranged from 3.81±0.42 to 4.09±0.82mmol/L (Table 4.59). It is apparent from the results that highest value (4.09±0.82mmol/L) was noticed in children served with doughnuts without supplementation followed by 3.98±0.63 mmol/L in group served with 20g/100g of sesame flour. Blood potassium is assessed to analyze concentration of potassium in the blood which may be in higher (hyperkalemia) or lower ranges (hypokalemia). 150 Table 4.57. Effect of protein enriched sesame fortified doughnuts on monocytes and eosinophils of school aged children Study intervals Monocytes (%) Means (Days) G1 G2 G3 S0 2.79±0.02 3.08±0.12 2.19±0.22 2.69±0.26 S30 2.67±0.34 2.89±0.38 2.14±0.16 2.57±0.17 S60 2.63±0.21 2.78±0.24 2.08±0.08 2.50±0.09 Means 2.70±0.13b 2.92±0.37a 2.14±0.25c Study intervals Eosinophil (%) Means (Days) G1 G2 G3 S0 2.39±0.02 2.19±0.13 2.22±0.24 2.27±0.12c S30 2.87±0.11 2.59±0.26 2.57±0.35 2.68±0.22b S60 3.33±0.25 2.76±0.34 3.08±0.46 3.06±0.37c Means 2.86±0.47a 2.51±0.34b 2.62±0.28b Means having same letters are statistically non-significant (P>0.05) Means± S.D 151 Table 4.58. Mean squares for serum electrolytes of school aged children SOV Df Blood sodium Blood chloride Blood potassium Intervals (D) 2 71.34* 14.90NS 0.04550NS Diets (T) 2 3.15NS 1.69NS 0.93800** D x T 4 5.80NS 3.95NS 0.00100NS Error 126 21.17 15.83 0.01999 Total 134 Df = Degree of freedom NS = Non-significant (P>0.05) * = Significant (P<0.05) ** = Highly significant (P<0.01) 152 Table 4.59. Effect of protein enriched sesame fortified doughnuts on blood sodium, chloride and potassium of school aged children Study intervals Blood sodium (mmol/L) Means (Days) G1 G2 G3 S0 138.32±1.23 138.09±1.00 138.23±1.03 138.21±0.62b S30 138.48±1.33 138.72±1.32 139.87±1.40 139.02±0.76ab S60 141.42±1.54 140.21±1.82 140.42±1.77 140.68±0.83ab Means 139.41±0.71 139.01±0.62 139.51±0.83 Study intervals Blood chloride (mmol/L) Means (Days) G1 G2 G3 S0 105.09±1.15 104.09±1.13 104.71±0.92 104.63±0.55 S30 105.18±0.99 104.79±1.02 105.39±1.12 105.12±0.63 S60 105.92±1.17 106.23±0.70 105.18±0.97 105.78±0.78 Means 105.40±0.83 105.04±0.56 105.09±0.67 Study intervals Blood potassium (mmol/L) (Days) Means G1 G2 G3 S0 4.07±0.03 3.77±0.09 3.94±0.13 3.93±0.20 S30 4.09±0.10 3.81±0.14 3.99±0.33 3.96±0.43 S60 4.12±0.23 3.84±0.32 4.01±0.42 3.99±0.62 Means 4.09±0.82a 3.81±0.42c 3.98±0.63b Means having same letters are statistically non-significant (P>0.05) Means± S.D 153 Potassium is an essential electrolyte with important role in cell metabolism. It also imparts it role in transmission of messages between muscles and nerves, functions in cardiac problems and contraction of muscles. The normal values of serum potassium in healthy individuals are 3.6- 5.5mmol/L (Pramina et al., 2013). 4.6.4.13. Renal function test (RFT) Mean squares for the influence of dietary intervention on renal function test (Table 4.60) revealed significant differences among the school children with respect to blood urea and serum creatinine due to experimental diets. However, there was non-significant impact of study intervals and interactions between diet and study intervals. whereas non-significant effect was found regarding study intervals as well as interaction between the study interval and experimental diets for these parameters. Means for blood urea (Table 4.61) showed maximum value (19.68±0.82 mg/dL) in the children served with 20g/100g of sesame flour followed by 10g/100g of sesame-based doughnuts (15.52±0.69mg/dL). The minimum value (15.15±0.40 mg/dL) was found in the group seved with doughnuts without supplementation. During study trial (60 days), consumption of sesame-based doughnuts did not show significant differences on the blood urea. Blood urea test is performed to analyze the amount of urea in a person’s blood which itself is derived from the waste products of urine. Breakdown of protein in body produces urea. Liver is the house for the production of urea and it is excreted out from the body through urine. Proper functioning of kidney is illustrated by this test. Blood urea level becomes high when kidneys are not working properly i.e. do not excrete it out from the body. Other causes for blood urea level increase are intake of high protein diet, heart failure and dehydration. The reference ranges of blood urea in health individuals are 11- 30mg/dL (Heise et al., 1994). Means for the serum creatinine (Table 4.61) exhibited maximum level (0.781±0.076mg/dL) in the children served with 20g/100g of sesame flour while the minimum level (0.731±0.024mg/dL) was found in the group nourished with doughnuts without supplementation. During 60 days intervention, serum creatinine was ranged from 0.750±0.035 to 0.763±0.067 mg/dL. This test is performed to analyze the level of creatinine in the blood. It is also done to ensure the proper working of kidneys in an individual. 154 Table 4.60. Mean squares for the renal function test of school aged children SOV Df Blood Urea Serum creatinine Intervals (D) 2 2.246NS 0.001905NS Diets (T) 2 284.481** 0.028185** D x T 4 0.596NS 0.000300NS Error 126 0.911 0.001006 Total 134 Df = Degree of freedom NS = Non-significant (P>0.05) ** = Highly significant (P<0.01) 155 Table 4.61. Effect of protein enriched sesame fortified doughnuts on blood urea and serum creatinine of school aged children Study intervals Blood urea (mg/dL) Means (Days) G1 G2 G3 S0 15.02±0.19 15.41±0.14 19.21±0.33 16.55±0.33 S30 15.21±0.27 15.52±0.32 19.72±0.48 16.82±0.55 S60 15.23±0.38 15.63±0.41 20.11±0.63 16.99±0.73 Means 15.15±0.40b 15.52±0.69b 19.68±0.82a Study intervals Serum creatinine (mg/dL) Means (Days) G1 G2 G3 S0 0.729±0.007 0.750±0.006 0.771±0.009 0.750±0.035 S30 0.732±0.009 0.758±0.018 0.781±0.026 0.757±0.055 S60 0.732±0.014 0.766±0.026 0.791±0.035 0.763±0.067 Means 0.731±0.024c 0.758±0.044b 0.781±0.076a Means having same letters are statistically non-significant (P>0.05) Means± S.D 156 Kidneys play an important role in reducing the creatinine level from body. Its elevated levels in blood may indicate improper functioning of the kidneys. The normal ranges of serum creatinine in healthy people are 0.59-1.2µg/dL (Heise et al., 1994). 4.6.4.14. Liver function test (LFT) Mean squares for the effect of protein enriched and sesame fortified doughnuts (Table 4.62) exhibited significant variations among the groups regarding total and conjugated bilirubin while non-significant impact of study intervals as well as interactions between intervals and experimental diets for all these traits. Means for the total bilirubin (Table 4.63) were maximum (0.720±0.035mg/dL) in the children consuming 10g/100g of sesame flour followed by the group served with doughnuts without supplementation (0.630±0.018mg/dL). During the whole study duration, total bilirubin was ranged from 0.623±0.004 to 0.650±0.023mg/dL. Means for the conjugated bilirubin (Table 4.63) depicted maximum value (0.458±0.023mg/dL) in the group served with 10g/100g of sesame doughnuts. At the baseline, the values were 0.370±0.034 mg/dL which were non-significantly decreased to 0.360±0.012 mg/dL after 60 days. The normal values oftotal and conjugated bilirubin in healthy persons are 0.1-1.2 and 0.0-1.6mg/dL, respectively (Thapa and Anuj, 2007). The mean results of unconjugated bilirubin (Table 4.63) described values ranged from 0.263±0.007 to 0.276±0.037 mg/dL among the children groups. Likewise, unconjugated bilirubin was slightly reduced from 0.279±0.035 to 0.262±0.015mg/dL at the end of intervention. Bilirubin is a substance (brownish yellow) present in bile which is formed by the breakdown of red blood cells in the liver. Afterwards, it is excreted through feces and contributes this color to the stool. Bilirubin has two forms in blood i.e. conjugated and unconjugated. Direct examination of blood reveals the total and conjugated bilirubin levels while unconjugated levels are estimated from both total and unconjugated levels in blood (Thapa and Anuj, 2007). 157 Table 4.62. Mean squares for liver function test of school aged children SOV Df Bilirubin Bilirubin Bilirubin Total Conjugated unconjugated Intervals (D) 2 0.008000NS 0.001125NS 0.003262NS Diets (T) 2 0.289500** 0.341045** 0.002222NS D x T 4 0.000500NS 0.000005NS 0.000432NS Error 126 0.002874 0.000706 0.001100 Total 134 Df = Degree of freedom NS = Non-significant (P>0.05) ** = Highly significant (P<0.01) 158 Table 4.63. Effect of protein enriched sesame fortified doughnuts on bilirubin (total, conjugated and unconjugated) of school aged children Study intervals Bilirubin total (mg/dL) Means (Days) G1 G2 G3 S0 0.640±0.011 0.740±0.007 0.570±0.019 0.650±0.023 S30 0.630±0.022 0.720±0.012 0.560±0.010 0.637±0.013 S60 0.620±0.005 0.700±0.006 0.550±0.007 0.623±0.004 Means 0.630±0.018b 0.720±0.035a 0.560±0.004c Study intervals Bilirubin conjugated (mg/dL) (Days) Means G1 G2 G3 S0 0.355±0.016 0.464±0.034 0.291±0.033 0.370±0.034 S30 0.351±0.010 0.458±0.025 0.286±0.022 0.365±0.021 S60 0.346±0.002 0.453±0.016 0.281±0.019 0.360±0.012 Means 0.351±0.015b 0.458±0.023a 0.286±0.003c Study intervals Bilirubin unconjugated (mg/dL) Means (Days) G1 G2 G3 S0 0.281±0.032 0.278±0.043 0.277±0.034 0.279±0.035 S30 0.276±0.022 0.274±0.039 0.263±0.029 0.271±0.024 S60 0.272±0.010 0.265±0.026 0.248±0.016 0.262±0.015 Means 0.276±0.037 0.272±0.024 0.263±0.007 Means having same letters are statistically non-significant (P>0.05); Means± S.D 159 CHAPTER 5 SUMMARY Malnutrition is the prime issue in developing countries. Pakistan is quiet young nations with a population of 207 million. The majority of the people from diverse age groups are hit by various forms of malnutrition including underweight, stunting, wasting, micronutrient deficiencies and array of communicable and non-communicable diseases. The women (pregnant & lactating) and children are the vulnerable segments of the population. It is one of the main cause of mortality and morbidity among the children. The current estimates suggest about 2-3% GDP (gross domestic product) loss linked with malnutrition. Food fortification, bio-fortification, dietary diversification and supplementation are globally adopted strategies to mitigate this menace. School age children are provided nutrition meals and snacks to fulfill partial requirements of energy and some essential nutrients. Snack foods are popular around the globe due to their convenience in availability, nutrient density, better satiety and shelf stability. These products can serve excellent vehicle for the delivery of which are considered best for growth and development. This instant activity has been planned to develop protein enriched and micronutrients fortified nutrient-dense doughnuts for school nutrition programs. After physico-chemical and nutritional assessment of the eight Pakistani white and black sesame cultivars, protein enriched (10-50 g/100g), micronutrient fortified doughnuts were prepared. Based on consumer acceptability, white sesame cultivar (TH- 6) and two supplementations levels (10g/100g and 20g/100) were selected for further use in study. Subsequently, selected treatments were subjected to biological evaluation using Sprague Dawley rats and later to probe their impact on the nutritional and biochemical profile of school children. The outcomes of the study are briefed as under: The proximate and mineral composition of sesame flours of white (TH-6, TS-5, TS-3, Til-89) and black (S-122, S-117, S-131, Latifi) cultivars revealed presence of moisture (7.23±0.32 to 11.82±0.38%), protein (33.59±0.08 to 38.97±0.28%), crude fat (10.25±0.52 to 17.23±0.29%), crude fiber (3.62±0.05 to 8.77±0.14%), ash (9.93±0.18 to 4.73±0.11%), NFE (22.75±0.52to 32.42±1.45%), sodium (29.24±0.82 to 60.12±0.89mg/100g), potassium (52.78±1.20 to 81.79±1.62mg/100g), calcium (46.82±0.55 to 80.21±1.90mg/100g), magnesium (33.45±0.95 to 59.23±1.48 mg/100g), zinc (7.23±0.16 to 19.53±0.36 mg/100g), iron (2.21±0.06 to 10.21±0.30mg/100g) and phosphorus (29.67±0.49to 63.47±1.11mg/100g), respectively. Means 160 for the amino acids profile in white and black sesame cultivars revealed existance of essential and non-essential amino acids like isoleucine (1.52±0.02 to 4.34±0.10g/100g), leucine (3.86±0.12 to 7.54±0.24g/100g g/100g), lysine (1.11±0.07 to 3.34±0.09g/100g), methionine (1.25±0.04 to 3.47±0.09g/100g), phenylalanine (2.24±0.05 to 4.48±0.06g/100g), threonine (2.55±0.07 to 4.38±0.12g/100g), tryptophan (0.81±0.01 to 2.57±0.06g/100g), valine (2.55±0.05 to 5.20±0.14g/100g) and histidine (1.83±0.05 to 3.10±0.08g/100g), alanine (0.78±0.02 to 5.27±0.14g/100g), arginine (1.87±0.08 to 4.83±0.09g/100g), aspartic acid (5.87±0.16 to 8.95±0.28g/100g), glutamic acid (12.23±0.22 to 18.67±0.20g/100g), glycine (1.19±0.02 to 5.96±0.13g/100g), cysteine (0.57±0.01 to 3.15±0.09g/100g), proline (0.75±0.03 to 3.85±0.07g/100g), serine (0.23±0.01 to 3.90±0.08g/100g) and tyrosine (1.82±0.05 to 4.25±0.07g/100g). Means for the antioxidants potential of white and black sesame flours of different cultivars exhibited total phenolic contents (1.56±0.025 to 7.32±0.22 mg GAE/g), 51.65±0.98 to 61.16±2.13% inhibition assessed through DPPH scavenging activity and 35.14±0.95 to 46.92±1.34 % inhibition as determined by using β-carotene bleaching method. Similarly, results for bioactive components of both white and black sesame confirmed varying degree of sesamin (1521.00±27.30 to 3996.00±129.90 ppm), sesamol (3121.00±86.60 to 4303.00±133.37ppm) and sesamolin (1532.33±31.89 to 3564.00±65.82ppm). Among the different cultivars, the maximum value of sesamin was observed in TH-6 (3996.00±129.90) followed by TS-5 (3753.00±120.67ppm) and TS-3 (3502.00±59.47ppm). It is apparent from the results that white cultivars of sesame contain more sesamin contents as compared to black cultivars. However, sesamol (4303.00±133.37ppm) and sesamolin (3564.00±65.82 ppm) concentrations were higher in S-122 (black cultivar). It is obvious from the results that sesamin and sesamol contents were higher in white sesame cultivars whereas sesamolin was more in black cultivars. White and black sesame flour supplemented doughnuts were evaluated for sensory attributes like color, aroma, taste, texture, chewability, mouthfeel and overall acceptability. It is apparent from the results that panelists appreciated doughnuts made from TH-6 (8.02±0.24) followed by TS-5 (7.79±0.18) and TS-3 (7.72±0.20) while Latifi (5.70±0.10) and S-133 (5.78±0.16) attained the lowest scores. It is clear from the results that doughnuts having white sesame flour were more liked by accessors due to better appearance, palatability, fine texture, chewiness, mouthfeel, and overall acceptability. This was probably linked with native liking for the white sesame in this part 161 of the world and the existence of some inherent constituents in black sesame seeds leading to slight bitterness in taste and blackish appearance of the product. Concerning various levels of supplementation, doughnuts having 10g/100g (7.67±0.24) to 20 g/100g (7.43±0.18) of either white or black sesame flours were more suitable due to delicious nutty aroma & flavor of sesame, mild brownish appearance, uniform texture, nutty mouthfeel and overall impression of the end product. With higher levels of supplementation, the color of doughnut become dark brown alongwith unpleasant nutty aroma, rubbery chewiness and non-uniform mouthfeel due to large pore size. Based on consumer acceptability and better-quality attributes, TH-6 (white sesame cultivar) with two supplementation levels (10g/100g and 20g/100g of sesame flour) selected for further development of doughnuts and assessed for nutritional contents, storage stability, calorific value, protein quality and feeding trial to see the impact of these products on serum biochemical and nutritional profile of school aged children. Mean values for moisture, protein, fat, fiber, ash and NFE were ranged from 7.53±0.19 to 8.38±0.12%, 22.69±0.31 to 24.85±0.40%, 9.25±0.23 to 14.41±0.46%, 1.52±0.08 to 2.27±0.33%, 0.90±0.11 to 1.84±0.36% and 49.10±1.45 to 68.45±0.90. Means for the selected minerals revealed that sodium, potassium, calcium, magnesium, phosphorus, iron and zinc contents were ranged from 42.03±0.95 to 65.35±0.74mg/100g, 19.65±0.46 to 32.52±0.71mg/100g, 84.26±1.70 to 236.38±3.00mg/100g, 95.06±2.31 to 176.21±3.10mg/100g, 385.27±7.78 to 520.18±8.80mg/100g, 10.25±0.15 to 22.64±0.64mg/100g and 10.15±0.23 to 20.92±0.24mg/100g, respectively. The results for textural attributes exhibited variations among the doughnuts with respect to hardness (42.27±0.81 to 113.72±3.64N), cohesiveness (0.15±0.21 to 0.25±0.42), springiness (0.71±0.26 to 0.77±0.41) and chewiness (0.23±0.18 to 0.47±0.02). Means for color i.e. L*, a* and b* values were ranged from 55.91±1.13 to 46.58±0.76, 15.68±0.31 to 9.56±0.21 and 36.42±0.93 to 18.50±0.25, respectively. Means for calorific value of doughnuts were ranged from 350.07±3.47 to 394.00±7.12Kcal/100g. Additionally, various treatments showed significant differences among the treatments for peroxide value (4.84±0.60 to 10.72±0.33 meq/kg), thiobarbituric acid number (0.426±0.16 to 0.658±0.30 mg/Kg), mold count (0.327±0.19 to 0.463±0.33×102CFU/g) and water activity (0.544±0.16 to 0.707±0.38). During 60 days storage, there was increase in water activity (0.597±0.27 to 0.657±0.45), POV (6.73±1.14 to 8.99±0.61meq/Kg), TBA No. (0.476±0.20 to 0.571±0.34 mg/kg) and mold count (0.258±0.23 to 0.503±0.46x102 CFU/g). Biological evaluation of selected formulations showed maximum protein efficiency ratio (2.43±0.29) and net protein 162 utilization (5.14±0.38) in rats fed on control diet i.e. soy protein whereas among the experimental diets, the highest PER (0.86±0.17) and NPU (2.81±0.25) was noticed in rats served with diet containing (20g/100g) sesame supplemented doughnuts (G2). Regarding nitrogen balance study parameters, maximum true digestibility (64.51±0.84%), biological value (69.64±1.08%) and net protein utilization (61.32±0.96%) in the diets containing 20g/100g sesame flour supplemented doughnuts. Overall, results for the growth and nitrogen balance index revealed that addition of sesame flour in snacks may improve the nutritional quality of these products and ultimately the consumers. After biological evaluation, selected treatments of doughnuts alongwith control were served to school children to find out their impact on nutritional biomarkers through the analysis of blood and urine. The results of anthropometric parameters exhibited values for height (143.86±0.76 cm to 144.84±0.91 cm), weight (37.58±0.07 to 38.30±0.23 Kg), BMI (18.03±0.35 to 18.32±0.43Kg/m2), head circumference (52.27±0.45cm to 52.61±0.53 cm) and mid upper arm circumference (19.42±0.23 to 19.62±0.35 cm). Study interval also had significant effect on weight (37.57±0.09 to 38.44±0.21kg) and BMI (18.03±0.17 to 18.34±0.38kg/m2) among the experimental groups. Overall, children served with sesame supplemented doughnuts depicted better growth as indicated by outcomes of height, weight and BMI. Biochemical results of the blood analysis depicted significant effect of sesame-based doughnuts on the serum ferritin (30.26±0.31 to 32.71±0.45 µg/L), serum zinc (9.74±0.09 to 10.11±0.22 µg/L), Hb (13.24±0.19 to 13.54±0.48 g/dL), RBC (4.56±0.23 to 4.67±0.44 Mill/Cu.mm), PCV (35.59±0.24 to 37.27±0.48 %), MCV (75.33±0.66 to 84.81±1.38 fL), MCH (28.07±0.20 to 30.51±0.47 Pg), and MCHC (38.37±0.24 to 38.68±0.49 %) of the school aged children. Group of children nourished with 20g/100 of sesame supplemented doughnuts showed pronounced changes in serum ferritin (28.60±0.15 to 34.31±0.36 µg/L), serum zinc (9.42±0.08 to 10.47±0.34 µg/L), Hb (12.75±0.16 to 13.96±0.35 g/dL), RBC (4.45±0.13 to 4.78±0.37 Mill/Cu.mm), PCV (34.33±0.18 to 38.13±0.37 %), MCV (76.47±0.74 to 84.37±1.42 fL), MCH (28.22±0.10 to 30.35±0.32 Pg) and MCHC (37.11±0.19 to 39.60±0.34 %) at the end of 60 days trial. Means for platelets (356800±1596 to 362033±2006 /Cu.mm), WBC (8866.1±69.7 to 9656.8±114.3 /Cu.mm), neutrophils (54.27±1.20 to 60.65±2.59 %), lymphocytes (36.62±0.39 to 38.28±0.59 %), monocytes (2.14±0.25 to 2.92±0.37 %) and eosinophils (2.51±0.34 to 2.86±0.47 %) remained within the normal ranges during the study. 163 The means for the serum sodium, chloride and potassium during the trial were ranged from 138.21±0.62 to 140.68±0.83mmol/L, 104.63±0.55 to 105.78±0.78mmol/L, 3.93±0.20 to 3.99±0.62mmol/L, respectively. The consumption of sesame flour supplemented and micro- nutrients fortified doughnuts were completely safe for the school children as evident from the results of kidney and liver function tests. Means for blood urea and serum creatinine showed maximum values of urea (19.68±0.82 mg/dL) and serum creatinine (0.781±0.076mg/dL) in the children served with 20g/100g of sesame flour. Likewise, mean values for the total bilirubin, conjugated- and unconjugated-bilirubin showed values ranged from 0.560±0.004 to 0.720±0.035, 0.286±0.003 to 0.458±0.023, and 0.263±0.007 to 0.276±0.037 mg/dL, respectively. The production of sesame-base doughnut is feasible and economical in Pakistan. The cost of per serving is low than that of competitive products like chocolate bars due to use of local raw materials. Additionally, thses producst are shelf stable and easy to prepare, handle, transport and store in tropical environment. The school children and other consumers have shown wider acceptability of sesame-based doughnut due to existing familiarity with sesame containg products like naan, rewary etc. Conclusively, the consumption of doughnuts containing 10-20g/100 sesame flour may deliver 140% additional protein, as well as % daily values of Zn and Fe. These products can be used for school nutrition programs especially in developing countries to reduce the severity of malnutrition. 164 RECOMMENDATIONS AND FUTURE DIRECTIONS Defatted sesame flour of white cultivars can be supplemented in wheat flour upto 20g/100g for the development of doughnuts without affecting consumer hedonic response. The flours should also be tested for the development of various other products like pan cake, pizza, rusks, leavened pan bread, biscuits as well as nutritious food bars. Consumption of 100g of sesame-based doughnuts can deliver 140% extra protein than that of conventional wheat-based doughnuts alongwith 100% daily value of iron and zinc. These protein enriched products should futher be tested by adding more micronutrients which are deficit in the community like vitamin A and D to curtail mineral and vitamin deficiencies through multiple nutrients food fortification. Nutrient-dense doughnuts should be used in school nutrition programs to reduce micronutrient deficiencies and protein energy malnutrition. These are ideal candidates for such inetrventions especially in developing countries due to cost-effectiveness, convenience in handling, wider consumer ecceptability, and sustainability due to use of local raw materials. Long-term efficacy trials with large sample size, gender variation, socio-economic background, and different age groups should be conducted to establish and validate the results of current intervention. Food multimixes comprising of locally cultivated raw materials should be developed and tested for range of products in different communities for sustainability of the diet-based interventions. Farmers should be encouraged for the cultivation of tradional crops like sesame and linseed alongwith modern commodities like quinoa, chia seeds etc. Likewise, food industry should develop novel functional food products by using these superfoods for better health of the consumers. 165 LIMITATIONS OF THE STUDY The availability of baselines data and local studies is a big challenge especially in developing counties. The sample size of existing national nutrition suvey is not sufficient for reliable and true representative results. Being high illitracey in the population, majority of the population has negligible information about the role of nutritious and balanced diet in optimum health, importance of personal hygiene and environmental sanitation. It is very difficult to get volunteers for efficacy trial even though food products are prepared in a hygienic environment and tested using animal model, assuming that there may be some harmful ingredients affecting their faith and sexual health. There available food composition tables or nutrient database is obsolete; hence, most of the time basic nutrient analysis is required before advanced analysis. This time consuming as well as wastage of available resources. Theere should be more exploration of locally grown crops for their nutritive and therapeutic potential so that those crops can be utilized in commercially available existing food products to enhance their nutritional quality as well as acceptability. The sample size for efficacy studies involving school children should be more to attain representative and accurate results. 166 LITERATURE CITED AACC (The American Association of Cereal Chemists). 2000. Approved Methods of American Association of Cereal Chemists, 10th Ed. 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Assoc. 1:800-806. 187 ANNEXURE-I Composition of Micronutrient Premix Per 100g Component Quantity Vitamin A (Palmitate) 4995IU Folic acid 0.4mg Niacin (Nicotinamide) 20mg Vitamin B1 (Thiamine) 1.5mg Vitamin B2 (Riboflavin) 1.7mg Vitamin B12 (Cyanocobalamin) 6mcg Vitamin C (Ascorbic acid) 70mg Zinc (Zinc Oxide) 10mg Iron (Ferric Pyrophosphate) 18mg Mixed Tocopherol 2.75mg Fortitech Inc. Schenectady, NY, USA 188 ANNEXURE-II 9-Point Hedonic Score System for Sensory Evaluation Name of Panelist: ______Designation:______Date: ______ Instruction for judges: 1. Evaluate for color by visual observation and for aroma by smelling 2. Chew the sample in mouth and score for taste, texture, chewability and mouthfeel 3. Before proceeding to the next sample, rinse mouth with water. 4. Do not disturb the order of the sample and do not consult with judges. Parameters Treatments for evaluation T0 T1 T2 T3 T4 T5 Color Aroma Taste Texture Chewability Mouthfeel Overall Acceptability Hedonic scale: 1. Disliked extremely 2. Disliked very much 3. Disliked moderately 4. Disliked slightly 5. Neither liked nor disliked 6. Liked slightly 7. Liked moderately 8. Liked very much 9. Liked extremely Remarks: ______ Signature: ______ 189 ANNEXURE-III .Institutional Biosafety/Bioethics Committee 190 ANNEXURE-IV Informed Consent Form Title of project: Probing the nutritive and therapeutic potential of Pakistani sesame cultivars for school going children Name of investigator: Ms. Sabiha Abbas I. Purpose of this research You are invited to participate your child in this research project to study the health effects of protein enriched and micronutrient fortified doughnuts. These snacks will help your child to get high protein which can help him to grow well as it is the main building block of our body. Moreover, it contains vitamin A, C, folic acid as well as minerals like iron, zinc and iodine which will help him to maintain good health, perform physical activity in a good way on daily basis, improved learning ability and protect him from deficiency of micronutrients. We are going to monitor their diet, growth, biochemical profile and overall health to find good connection between healthy snacks and improved growth and development. II. Procedure Once we get approval from your side, your child will be examined by the medical officer for good health to participate in the study. Different growth parameters like height, weight, body mass index (BMI) and mid upper arm circumference (MUAC) will be measured. Selected children will be then checked for hematological studies such as complete blood count (CBC), electrolytes balance, renal function test (RFT), liver function test (LFT), serum ferritin and serum zinc. For all these tests, 5mL of blood sample will be drawn. All blood sampling will be done during the school hours and lab technician will come to school for getting the blood samples. International standards of hygiene will be adopted during this procedure. Based on the reports of blood analysis children will be screened according to the set criteria of inclusion and exclusion. When your child become participant of our study, a day will be decided (will be informed you later) when you will have to come and provide us medical history of your child and give answers to some of our questions related to your living. Duration of whole study is two months, during this interval your child will be given fixed portion of snacks daily during the mid-day break. Constant supervision for his health and diet will be done by the experts. Once it all set the follow up blood testing will be done after 30 days interval. Meal supervision will be done daily and investigation about anthropometric measurements will be carried out at every week interval. III. Risks There are no more than minimal risks for participating in this study. In some individuals a bruise is formed during blood collection; however, certified phlebotomists will draw blood to prevent bruise formation. Some individuals may faint during blood drawing. If child is fainted, he will be let for rest as for as you want in comfortable position and environment. Starchy foods sometime cause intestinal discomfort in some people. If you feel stomach discomfort, please inform your investigator. If you have any food allergy, please inform your investigator. Sesame and wheat are the main ingredients in the snacks. 191 IV. Benefits Participation in this study will provide valuable information about the potential health benefits of healthy snacks. It will also help elucidate the health benefits connected with the consumption of high protein and fortified nutrient dense food. Complete medical examination and clinical reports will be shared which will be beneficial to pin point growth and development of the child and micronutrient status of the children. V. Extent of anonymity and confidentiality Information will be kept strictly confidential. Subjects will be assigned code numbers and they will not be identified by their names. Individual subjects will be referred by a code number for data analysis and publication of the results. Results and data sheets will be placed in the locked cabinet to maintain the confidentiality. Code numbers will be kept in separate locked cabinet. VI. Freedom to withdraw Study participation will be fully free. There may be reasons under which investigator may determine you should not participate in this study. If you have food allergy asked to refrain from participating. VII. Subject’s responsibilities Be punctual during the study interval Consume the snacks as instructed by the instructor Allow for measurements to be taken (weight, height, mid upper arm circumference) Allow for blood drawing at 0, 30 and 60 days of study Communicate his daily diet for the completion of diet record VIII. Subject’s permission I have read the consent form and conditions of this research project. I have had all my questions answered. I hereby acknowledge the above and give my voluntary consent: ------Date: ------Subject’s guardian signature Subject Information Printed name: ------Address: ------. I would like a copy of my child’s body measurements . I would like a copy of my child’s nutrient analysis . I would like a copy of all blood biochemical tests . I would like to have dietary consultation for my child’s health at the end of the study Should I have any pertinent questions about this research or research subject’s right and whom to contact in the event of a research related injury to the subject, I may contact: Sabiha Abbas, Ph.D Scholar, Investigator +92 334 7130642 Dr. Mian Kamran Sharif, Supervisor +92 333 8608341 192 ANNEXURE-V Subjects Distribution for Efficacy Trial Consent form received N= 85 Screening for height & weight N= 60 Age 9-12 years Exclusion Criteria* Randomization N=48 Withdrawn (N=3) Reason: Left the school before the start of study G1 (N=15) G2 (N=15) G3 (N=15) Fortified Doughnuts Fortified doughnuts with Fortified doughnuts with without supplementation 10g/100g of sesame flour 20g/100g of sesame flour 193