ASSESSING THE POTENTIAL OF HOUSEHOLD FOOD PROCESSING TO IMPROVE ZINC NUTRITION IN MALAWI AN ABSTRACT SUBMITTED ON THE ELEVENTH OF JULY 2016

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1 ASSESSING THE POTENTIAL OF HOUSEHOLD FOOD PROCESSING TO IMPROVE ZINC NUTRITION IN MALAWI AN ABSTRACT SUBMITTED ON THE ELEVENTH OF JULY 2016 TO THE PAYSON GRADUATE PROGRAM IN GLOBAL DEVELOPMENT IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE TULANE UNIVERSITY LAW SCHOOL FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY

2 ABSTRACT Malawi is one of the least-developed countries in sub-saharan Africa, with high rates of food insecurity, stunting, and micronutrient malnutrition. Zinc deficiency is associated with a number of health problems in Malawi, including diarrhea, pneumonia, stunting, and adverse pregnancy outcomes. Maize is the staple of the national diet, yet the zinc nutrition of maizebased diets is compromised by the presence of phytate, a potent inhibitor of zinc absorption. Phytate levels can be reduced by basic household processing methods such as soaking, germinating, and fermenting, thus increasing the rate of zinc absorption. Novel research on sustainable approaches to addressing malnutrition using these kinds of food-based methods is urgently needed. Using food consumption data from the Malawi Third Integrated Household Survey and the latest models to predict zinc absorption, this study estimates the proportion of the population at risk of zinc deficiency, with a focus on vulnerable sub-groups including women and children. Next, it uses a simulation model to estimate the effects of reducing dietary phytate through processing and compares those results to an alternative simulation based on biofortification. Finally, this study examines the practical considerations necessary to promote improved maize processing using a behavior change communication approach and estimates the costeffectiveness of the intervention compared to alternatives. The study s findings indicate that the initially high proportion of people at risk of zinc deficiency in Malawi can be substantially reduced by processing maize to reduce phytate.

3 Compared to biofortification, the impact of processing was greater for all regions and subgroups, and the advantage of processing was more pronounced in the South and in rural areas. An intervention to promote these improved methods using behavior change communication and nutrition education compares favorably against alternatives on a cost-effectiveness basis. A thorough analysis of culture and gender norms, the decision-making context, and the drivers of food choice in Malawi suggest that an intervention to promote household-level maize processing can be culturally appropriate and scalable if the context is properly considered. Given these findings, food-based approaches such as household level food processing should be given greater attention in policy and practice to sustainably improve food security and health outcomes.

4 ASSESSING THE POTENTIAL OF HOUSEHOLD FOOD PROCESSING TO IMPROVE ZINC NUTRITION IN MALAWI A DISSERTATION SUBMITTED ON THE ELEVENTH OF JULY 2016 TO THE PAYSON GRADUATE PROGRAM IN GLOBAL DEVELOPMENT IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE TULANE UNIVERSITY LAW SCHOOL FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY

5 Copyright by Gregory M. Sclama, 2016 All Rights Reserved

6 TABLE OF CONTENTS 1. Introduction The Research Problem Purpose Statement and Significance Background and Literature Review Malnutrition, Health, and Development Micronutrient Malnutrition: Dietary Diversity, Nutrient Density, and Bioavailability Overview of Zinc Nutrition Strategies to Improve Zinc Nutrition: Household Food Processing Strategies to Improve Zinc Nutrition: Alternatives to Household Processing Malawi: Country Overview and Study Population Background Conceptual Framework for Research Research Questions Methodology Assessment of Prevalence of Zinc Malnutrition in Malawi and Simulation of the Impact of Food Processing and Biofortification Data Sources, Cleaning, and Preparation Preparation of Malawi Food Composition Table Calculation of Zinc Nutrition Indicators and Total Absorbed Zinc Calculation of the Prevalence of Inadequate Zinc Intakes Simulation of Household Food Processing Simulation of Biofortification Validity and Limitations of the Methodology Primary Research Findings: Simulation Results Research Question Research Question ii

7 6.3 Research Question Implications of the Findings of this Study for Development Programming to Reduce Malnutrition in Malawi: Regional Prioritization Discussion: Sensitivity Analysis and Effect of Methodological Decisions Additional Research Findings: Cost Considerations for Programming Introduction: Disability Adjusted Life-Years (DALYs) Economic Quantification of the Burden of Zinc Deficiency in Malawi Comparison of Cost-Effectiveness of Alternative Zinc Interventions Discussion of Cost Considerations for Programming Additional Research Findings: Considerations for Successful Implementation The Applied Behavior Change Model Sustainability and Scalability Conclusion Summary Significance Future Research Needs Ethical Concerns Appendices: Full Results from Simulations A) Baseline Analysis B) Household Level Processing of Maize - 100% coverage C) Household Level Processing of Maize - 40% coverage D) Household Level Processing of Maize - 20% coverage E) Household Level Processing of Maize - 10% coverage F) Biofortified Maize - 100% Coverage G) Biofortified Maize - 40% Coverage H) Biofortified Maize - 20% Coverage I) Biofortified Maize - 10% Coverage References iii

8 LIST OF TABLES Table 1: Zinc Mean Physiological Requirements (MPR), IZiNCG 2004: Table 2: Zinc, Phytate, and Phytate:Zinc Molar Ratios of Several Staple foods from Malawi and Ghana (Source: Gibson and Ferguson, 2008; Gibson, 2012) Table 3: Comparison of WHO and IZINCG Estimates of Zinc Absorption Based on Phytate:Zinc Molar Ratios. Source: IZINCG, Table 4: Selected Fermented Foods (Sources: Steinkrauss in Mensah and Thompkins; Aworh 2008; Ibnouf 2012 ) Table 5: Summary Table of Studies of Processing on Maximum Percent of Phytate Reduction.. 31 Table 6: Select indicators for zinc nutrition for Malawi, Zambia, Ghana, and regional estimates. (Source: IZiNCG, 2004; Wuehler et al., 2005) Table 7: Number of Household Members Belonging to Each Group, by Region and Urban Status Table 8: Examples of Food Categories and Foods Survey in the Malawi Third Integrated Household Survey Table 9: National Indicators of Zinc Nutrition for Females Table 10: National Indicators of Zinc Nutrition for Males Table 11: National Indicators of Zinc Nutrition for Children Table 12: Indicators of Zinc Nutrition, Regional and Urban Disaggregation Table 13: Regional Comparisons of Zinc Deficiency Prevalence Table 14: Population Mean Molar Ratio at Baseline Compared to Various Levels of Coverage of Household Processing Table 15: Prevalence of Zinc Deficiency Risk for Women, Comparison of Household Processing at Various Levels of Coverage (20% coefficient of variation) Table 16: Prevalence of Zinc Deficiency Risk for Men, Comparison of Household Processing at Various Levels of Coverage (20% coefficient of variation) Table 17: Prevalence of Zinc Deficiency Risk for Children Age 9-13, Comparison of Household Processing at Various Levels of Coverage (20% coefficient of variation) iv

9 Table 18: Molar ratios at various levels of intervention coverage, along with the percentage change from baseline Table 19: Regional Prevalence Rates of Zinc Deficiency Risk Resulting from Household Level Maize Processing at Various Levels of Coverage for Women Table 20: Men and Children: Proportion of Population with Absorbed Zinc below their MPR Table 21: Phytate-to-zinc Molar Ratio at baseline and under food processing and biofortification scenarios Table 22: Prevalence of Risk of Zinc Deficiency at Various Coverage Levels for Select Groups Table 23: Prevalence Rates of Zinc Deficiency Risk for Women >14 years, assumed 20% coefficient of variation Table 24: Regional Summary Table with Program Implications Table 25: Effect on percentage falling below the MPR from changes to the assumption of the CV. Two groups represent one in which the mean exceeds the MPR, and one in which the mean falls below the MPR Table 26: Percent of the Adult Woman Population below MPR: Effect of Using the IOM cutpoint instead of the IZiNCG cut-point Table 27: Data Used to Calculate DALYs for Zinc Deficiency Table 28: Years of Productive Life Lost due to zinc Deficiency in Malawi Table 29: Yearly costs of Biofortification of Maize for Provitamin A. (Source: Meenakshi et al., 2010.) Table 30: Comparison of Cost-Effectiveness of Interventions to Address Zinc Deficiency Table 31: Highest Level of School Attended by Women over 18 years, % Women LIST OF FIGURES Figure 1: Zinc Deficiency by Region (Source: Wessells, K. R., & Brown, K. H. (2012) Figure 2: Malawi Location in Africa and Districts and Regions of Malawi Figure 3: Conceptual Framework for Research Figure 4: Prevalence of Inadequate Zinc Intake, Women, Regional Figure 5: Estimated Prevalence of Inadequate Zinc Intake for Females Age 14-18, at Baseline and After Household Maize Processing Intervention at Various Levels of Coverage v

10 1 1. Introduction 1.1 The Research Problem Despite global progress to combat hunger, micronutrient malnutrition remains a tremendous burden in the developing world. Micronutrient deficiencies affect nearly one-third of the population in the developing world, with a significantly greater burden on the poor. Addressing malnutrition is a priority for achieving the post-2015 Sustainable Development Goals, and innovative approaches to food security programming and policy are urgently required. Zinc is an essential trace element involved in a wide range of biological functions including growth and protection against infections. Widespread zinc deficiency contributes to high rates of stunting and disease risk in the poorest countries. In Malawi, previous estimates based on national food supply have found that 34% of the population is at risk of inadequate zinc intakes, while local studies have found deficiency rates as high as 90% (IZiNCG, 2004; Siyame et al., 2013). For much of the developing world, staple cereal crops are the primary source of energy and micronutrients. In Malawi, one of the poorest countries in the world, maize contributes over 60% of total energy and zinc to the diet (Tinna et al., 2013). Although maize contains high levels of zinc, the bioavailability of zinc in maize is limited due to the presence of antinutrients which inhibit absorption. The most potent inhibitor of zinc absorption is phytate, a form of phosphorous present in very high amounts in many plant foods. Staples such as maize can be processed before consumption to reduce phytate levels and to increase the bioavailability of zinc (IZiNCG, 2004). Nevertheless, household level food processing practices which reduce phytate, such as soaking, germination, and fermentation, are not widely practiced in

11 2 Malawi. Moreover, the promotion of improved household food processing methods has been largely overlooked as a viable intervention for nutrition and food security programming, which have mostly focused instead on supplementation and fortification, and more recently, biofortification. Despite recognition that household processing practices can reduce phytate levels and increase nutrient availability in staple diets, there is very limited evidence on the degree to which they can reduce the prevalence of micronutrient deficiencies for national populations and vulnerable subgroups. An evidence base is urgently needed to evaluate this potential and to consider promotion of household processing as a viable intervention and policy option for addressing malnutrition. 1.2 Purpose Statement and Significance The purpose of this study is to quantitatively estimate the reduction in the population prevalence of zinc deficiency that could be achieved through the promotion of improved maize processing in Malawi. It is also the purpose of this study to compare these estimates to those resulting from an alternative program of biofortification, and to examine the costs, barriers, and programmatic considerations that need to be made to successfully promote these processing methods in Malawian communities if warranted by the evidence. These objectives were accomplished by conducting a dietary assessment of population baseline zinc deficiency in Malawi, followed by a simulation to model the effect of reducing phytate levels through food processing. Using nationally representative data from the Malawi Third Integrated Household Survey, this study provides a more accurate estimation of the extent of zinc deficiency for vulnerable subgroups than currently exists in the literature. By simulating the impact of household food processing on the nutritional content of maize, this study estimates the change in the prevalence of zinc deficiency that could be achieved through promoting these improved techniques. A comparison between household processing and biofortification at various levels of

12 3 coverage reveals the relative benefits of each intervention in different geographical areas of Malawi. By analyzing the feasibility, benefits, and challenges to promotion of these methods, this research contributes to the knowledge base, provides a roadmap for programming, and forms a baseline for future research. The following research questions were answered through this study: Research Question 1a: What is the current estimated prevalence of zinc deficiency in Malawi for population subgroups of interest? Research Question 1b: How do the estimated prevalence levels compare across regions and between urban and rural residence? Research Question 2a: What is the expected reduction in the prevalence of zinc deficiency resulting from the adoption of improved household maize processing techniques? Research Question 2b: How do these changes vary across regions and between urban and rural residence? Research Question 3a: What is the expected reduction in the prevalence of zinc deficiency from an alternative intervention based on biofortification? Research Question 3b: How does an intervention to promote improved food processing compare to biofortification at the national level at various levels of coverage? Research Question 3c: How does an intervention to promote improved food processing compare to biofortification for each geographical region?

13 4 This research is important and timely for several reasons. First, there is an urgent need for evidence on sustainable, food-based approaches to combat malnutrition, and this study provides such evidence (Ruel, 2001). This type of household-level intervention can reduce reliance on outside resources and increase the utilization of local foods for sustainable, long term improvements in nutrition. In addition to providing evidence for an alternative approach to food security programming, this study contributes to the literature specifically on combatting zinc malnutrition in Malawi and throughout sub-saharan Africa. The International Zinc Consultative Group has called for greater attention to the design and evaluation of the nutrition and health impacts of dietary interventions to increase zinc status, which include food processing to reduce phytate content (Brown and Hess, 2009). This dissertation provides an original contribution to the practical and theoretical knowledge and evidence base about how innovative nutrition programs can sustainably address chronic malnutrition to ensure long term development.

14 5 2. Background and Literature Review 2.1 Malnutrition, Health, and Development The Burden of Malnutrition Despite substantial progress, 868 million people remain undernourished worldwide (FAO, 2013). Twenty percent of the children under-5 in the developing world are underweight while nearly a third are stunted (Black et al., 2008). Malnutrition underlies roughly half of deaths of all causes in children under five (Black et al., 2003). The conditions of malnutrition may be caused not only by inadequate protein or caloric intake, but also by micronutrient deficiency (Levinger, 1995). An estimated two billion people suffer from deficiencies in micronutrients such as zinc, iron, iodine, and vitamin A, which cause death in childbirth, irreversible damage to children during early years of growth, and prevents adults from working (FAO, 2013). The Relationship between Malnutrition and Disease A review in The Lancet of the causes of under-5 mortality found that while causes vary substantially from one country to another, undernutrition is a key underlying cause of child deaths associated with infectious disease (Black et al., 2003). The analysis revealed that underweight and micronutrient malnutrition are major risk factors: 53% of all child deaths from the studies examined could be attributed to being underweight (Black et al., 2003). Other studies confirmed that this percentage ranges from 44.8% because of measles to 60.7% from diarrhea (Caulfield et al., 2004). Acute malnutrition is strongly associated with prevalence of HIV and tuberculosis (Lazzerini et al., 2013). In developing countries where malnutrition and infection are both high, successful control of them requires actions directed at both (Scrimshaw et al., 1968).

15 6 The Importance of Nutrition for Development Good health and nutrition are essential elements of sustainable development as defined through the lenses of economic growth and a capabilities approach to development. The relationship between nutrition and health is well established (Fogel, 2004). Health and human capital are both inputs and output of the aggregate production function (Todaro and Smith, 2012). As an input, good health is a prerequisite for successful development (Todaro and Smith, 2012). Good health is an output of development through increased incomes, improved access to health care and nutritious foods, and informed choices. Good health is also a critical component of development as viewed through a capabilities approach. In this sense, development is also an expansion of the freedoms that one values (Sen, 2000). The freedom to avoid starvation, under-nourishment, morbidity, and premature mortality, as well as the freedom to enjoy the social and cultural functions of food are viewed intrinsically as ends in themselves. Nutrition and food security therefore are essential components of the intrinsically important ability to live a healthy life free of deprivation. Malnutrition too, is a matter of social justice and equity the prevalence of malnutrition is often many times higher among those who are socioeconomically deprived within a given geographic area (Lazzerini et al., 2013). The Relationship between Nutrition and Economic Growth Childhood malnutrition has significant consequences for lifetime achievement and earnings. A review of a wide body of evidence across different settings links poor nutrition in childhood to negative outcomes in intellectual development (Grantham-McGregor et al., 1999). Studies in the Philippines and in Kenya found a relationship between child nutrition statuses and test scores (Glewwe et al., 1996; Bhargava, 1996). These negative effects from childhood malnutrition persist over the lifetime with consequences for economic productivity. In an exhaustive review of the

16 7 literature on the relationship between health, nutrition, and economic development, Strauss and Thomas (1998) concluded that the evidence supports a positive relationship between nutrition and increased lifetime wages, and that poorly nourished people were more likely to be unemployed. The Lancet examined five long-standing studies for the relationship between maternal and child undernutrition and adult outcomes (Victora et al., 2008). The authors found strong associations between undernutrition and adult height, reduced years of schooling, reduced economic productivity, and low offspring birthweight. Many of these negative outcomes are permanent and intergenerational. The authors concluded that, damage suffered in early life leads to permanent impairment and might also affect future generations. Its prevention will probably bring about important health, educational, and economic benefits (Victora et al., 2008). In addition to lifelong consequences of damage suffered during early childhood, there is evidence for the intergenerationality of malnutrition. Undernourished mothers are more likely than well-nourished mothers to give birth to low birthweight children, a condition which is associated with increased risk of death in the first year of life (Overpeck et al., 1992). Malnutrition leads to direct economic losses through reduced physical capacity and increased health care costs, and indirectly through losses due to poor cognitive development and schooling (Lazzerini et al., 2013). Micronutrient deficiencies are estimated to cost both India and China each at least $2.5-5 billion annually. The World Bank Estimates that in Sierra Leone iron deficiency anemia affecting the productivity for the female labor force was estimated to cost over $94.5 billion dollars in a five-year period. Preventing one child from being born underweight is estimated to be worth $580 (Shekar et al., 2006). Interventions that address malnutrition may reduce poverty and lead to national economic growth.

17 8 Food Security and Nutrition as a Development Priority: Historical Context Food security has long been recognized as a priority for development. The 1974 World Food Conference brought international attention the issues of global food security and asserted a universal right of people to be free from hunger and malnutrition (Shekar et al., 2006). The 1996 Rome Declaration restated the political consensus to reduce the number of malnourished people in the world, defining food security as the state in which, all people, at all times, have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life (World Food Summit, 1996). Freedom from hunger and malnutrition are central components of both the Millennium Development Goals and the Sustainable Development Goals. The nutritional paradigm through which the problems of food security have been viewed has evolved over time from a focus on protein-energy malnutrition to the prioritization of micronutrients that we see today (Semba, 2011). According to Allen (2000), protein deficiency was considered the single most important aspect of malnutrition by the World Health Organization and Food and Agriculture Organization until about Kwashiorkor, a manifestation of severe protein malnutrition characterized by edema, was common in rural Africa where populations used primarily starchy staples as infant weaning foods. Over the following decade or so, empirical evidence suggested that the role of protein as a limiting nutrient had been overstated, and that few diets worldwide could actually be considered deficient in protein. The role of total caloric intake in the diet was then increasingly recognized as important, and a paradigm of protein-energy malnutrition was then adopted. Nevertheless, the importance of micronutrients was largely overlooked in this period, as the focus on caloric deficit coincided with a push to increase agricultural production, principally of cereal staple foods. The technological advances of the so-called green revolution led to tremendous

18 9 increases in yields of crops, increasing the total quantity of food available for consumption, but neglecting concerns about dietary quality or micronutrients. In the 1980s, the concept of food security largely shifted from aggregate production at the international and national level to the household level. This increased focus on the household level grew out of the recognition that expanded food production in an area would not necessarily allow all households and family members to meet their food needs (Eicher and Staatz, 1990). Amartya Sen s (1981) analysis of historical famines brought attention to the fact that people starved when there was no aggregate food shortage. Throughout the 1980s, research began to shed light on the fact that dietary quality, including intake of animal products, was an important predictor of health outcomes (Allen, 2000). The role of deficiencies of certain micronutrients in common functional outcomes in the developing world were established; these included the role of iron deficiency in anemia, iodine in goiter, and Vitamin A in preventable blindness. Consensus surrounding the importance of micronutrient malnutrition was galvanized in the 1990 World Summit for Children, led by UNICEF, the World Bank, and the World Health Organization. The Summit established a commitment to reduce iodine, iron, and Vitamin A deficiencies over the next two decades, and provided a framework for action during that period. One of the outcomes of that meeting was the establishment in 1992 of the Micronutrient Initiative, an organization that advises governments on strengthening the delivery systems specifically for high-priority vitamins and minerals. The following years saw a great deal of research that both underscored the role of multiple micronutrient deficiency in child morbidity and mortality, and supported the conclusion that micronutrient interventions were some of the most cost-effective interventions for addressing child well-being (World Bank, 1993). The Copenhagen Consensus advises governments and donors on the most cost-effective use of funds for aid and development. In 2004 the Copenhagen Consensus ranked nutrition interventions first among 17 potential development investments for their potential

19 10 to generate returns, above trade liberalization, malaria prevention, and sanitation (Shekar et al., 1996). The Consensus identified supplementing vitamins for malnourished children as one of the world s best investments. The centrality of reducing malnutrition the in Millennium Development Goals underscores the importance of nutrition and food security as a development priority. In addition to being explicitly stated in the first goal of Eradicating Extreme Poverty and Hunger, nutrition is implicit to a number of other goals including reducing child mortality, improving maternal health, achieving primary education, and combatting diseases (UNDP, n.d.). Malnutrition negatively affects school attendance and achievement (Goal 2), is directly associated with child deaths (Goal 4) and maternal health (Goal 5), and increases the risk and disease burden of HIV and other diseases (Goal 6). Nutritional wellbeing is a fundamental component of the post-2015 Sustainable Development Goals, as Goal 2 specifically seeks to end hunger and end all forms of malnutrition (Webb, 2014). There is no doubt that historical efforts to reduce hunger and malnutrition have made progress. The Food and Agriculture Organization estimates that since 1990, the prevalence of undernourishment in developing countries has dropped from 23% to 15%, and the prevalence of stunting has declined from 44.6% to 28% (FAO, 2013). Nevertheless, micronutrient malnutrition remains incredibly high, affecting an estimated 2 billion people worldwide (FAO, 2013). The persistence of widespread micronutrient malnutrition is contributing to failed progress toward internationally established development goals (Shekar et al., 2006). Progress, especially in reducing neonatal and maternal mortality, has been extremely slow with little progress in some regions such as Sub- Saharan Africa. Future progress requires continued efforts to address multiple micronutrient deficiency using sustainable, food-based approaches.

20 Micronutrient Malnutrition: Dietary Diversity, Nutrient Density, and Bioavailability Introduction to Micronutrient Malnutrition Malnutrition may be caused by micronutrient deficiency rather than simply by inadequate protein or caloric intake (Levinger, 1995). Micronutrient deficiencies are estimated to affect nearly one third of the population in the developing world, with a significantly higher burden on the poor (Shekar et al., 2006). The causes of micronutrient malnutrition are complex, including economic aspects such as lack of access to healthy foods, behavioral aspects such as poor feeding and care practices, low levels of sanitation, and gendered aspects such as lack of women s time. Micronutrient malnutrition is often referred to as hidden hunger, because its effects, while less apparent than severe underweight, are extremely pervasive and pose a significant burden. Micronutrients include vitamins and minerals. Vitamins are compounds containing nitrogen and carbon that are essential for physiological function (Hübner and Arendt, 2013). Because they are not synthesized by human metabolism, they must be obtained in the diet. Minerals such as zinc, iron, and calcium, are naturally occurring inorganic substances essential for body function and for maintaining osmotic pressure in the body (Hübner and Arendt 2013). Poor Populations Have Low Dietary Diversity Dietary diversity is an important factor for nutritional status. Research supports the strong link between dietary diversity indicators and household level food security. Measures of dietary diversity have been found to have strong correlations with increased caloric intake across urbanrural settings and across seasons in diverse country settings (Hoddinott and Yohannes, 2002). A review of studies of eleven Demographic and Health Surveys (DHS) confirmed the efficacy of dietary diversity scores (DDS) as an indicator of nutritional status, finding a strong bivariate association between DDS and height-for-age Z-scores (Arimond and Ruel, 2004).

21 12 However, poverty constrains access to diverse, nutrient-dense foods, and poor populations are often dependent on a small number of staple foods for most of their energy and nutrients. Many people in developing countries cannot afford vegetables, meat, dairy, eggs, legumes, and fish products, which provide essential nutrients. Staples, which may include cereals such as maize or starchy tubers such as cassava, are less nutrient-dense and have anti-nutritional factors that affect the absorption of nutrients. Small additions of animal-based products or simple, traditional processing methods can substantially increase micronutrient availability (Perlas and Gibson 2005; Mensah and Thompkins, 2003). Staple Foods Have Low Nutrient Density The World Health Organization recommends that children be exclusively breastfed for the first 6 months of life (Dewey, 2003). Exclusive breastfeeding provides all essential nutrients, provides the baby with a food supply that is not competitive with rest of family, and ensures that the food supply is not contaminated. However, for the majority of children in the developing world past the age of exclusive breastfeeding, cereal crops are the main sources of energy, as well as protein, zinc, iron, niacin, and riboflavin (Hotz and Gibson, 2001). Between the ages of 6 and 12 months, young children require foods with a very high nutrient density, especially nutrients zinc and iron (Dewey, 2013). However the staple foods on which young children rely are often low in total micronutrients low in micronutrient bioavailability due to the presence of inhibitors of absorption. Additionally cereals are often made into gruels, which must be diluted with water before children can consume them, which both decreases the nutrient density of the foods and introduce an opportunity for pathogenic contamination (Brown et al., 1989; Motarjemi et al., 1993; Kimmons and Brown, 1999).

22 13 Micronutrient Bioavailability Is Low in Cereal Staples Zinc, iron, and calcium are defined by the WHO as problem nutrients, and their concentrations in complementary foods often falls below the recommended intake levels. In addition, the bioavailability of each of these minerals is inhibited by anti-nutrients found in staple foods. Bioavailability can be defined as the proportion of an ingested trace element in food that is absorbed and utilized for normal metabolic and physiological functions or storage (Gibson et al., 2006). Bioavailability is influenced by numerous factors both related to the diet and to the consumer; these include the chemical form of the nutrient, the nature of the food matrix, interactions between nutrients and other components within food, and the treatment of food during processing (Gibson et al., 2006). 2.3 Overview of Zinc Nutrition Zinc is an essential trace element, a micronutrient that must be obtained from the diet, and it is found in numerous plant and animal-based foods. Zinc plays many biological roles for plants, animals, and humans. Zinc is an essential nutrient for human nutrition, and is known to be involved in the activity of over 300 enzymes, as well as a wide range of biological functions (Gibson, 2012). As the body does not maintain functional reserves of zinc, a regular dietary zinc supply is essential. Because zinc is essential for cell division, protein synthesis, oxygen transport, growth, and protection against infections, infants, children, adolescents, and pregnant woman are at highest risk form deficiency, although adults and elderly also suffer from the consequences of zinc deficiency (WHO, 2004; Michaelsen et al., 2009). The Burden of Zinc Deficiency The Lancet series on maternal and child undernutrition found that zinc deficiency is responsible for 4% of the global burden of mortality and morbidity in young children (Black et al., 2008). Zinc deficiency contributes to poor growth in young children (stunting), low weight-for-age, and

23 14 increased disease risk, and poses one of the largest disease burdens for child undernutrition (Caulfield et al., 2004; Black et al., 2008). Zinc deficiency increases the risk of mortality from diarrhea, pneumonia, and malaria by 13-21% (Black et al., 2003). Zinc nutrition is also important for preventing low birthweight, one of the causes of asphyxia, and to prevent diarrhea, malaria, and other nutrition-related disorders, which are major causes of newborn death in Malawi and Sub- Saharan Africa. Zinc is even more important for low-birthweight infants and children that are severely malnourished. In adolescents, zinc deficiency is associated with height-for-age, weight, bone development, anemia, enlargement of the spleen and liver, and delayed sexual maturation (Halsted et al., 1972). The effects of zinc supplementation on linear growth and stunting, diarrhea prevalence, and serum zinc concentrations have been documented, and zinc supplementation was rated one of the best interventions for reducing malnutrition in the Copenhagen Consensus (Bhutta et al., 1999; Brown et al., 2002; Denova-Gutierrez et al., 2008; Brown and Hess, 2009). In maternal health and pregnancy outcomes, zinc status has been associated with intrauterine growth retardation, low-birthweight, poor fetal neural development, preterm delivery, pregnancyinduced hypertension and increased neonatal mortality (IZiNCG, 2004). Other negative consequences for maternal health have been observed, such as prolonged labor, placental abruption, premature rupture of membranes, preeclampsia, hemorrhage, and postpartum malnutrition and infection. The World Health Organization suggests that maternal zinc nutrition may affect child neurological development and immunocompetence over the entire course of the child s life (Osendarp et al., 2001; Caulfield et al., 2004). It is believed that poor maternal zinc status affects postnatal development including poor infant growth and increased risk of infection, however the evidence base suffers from a lack of randomized-controlled studies and trials in low-income countries

24 15 (IZiNCG, 2004). Girls and young women who suffer zinc deficiency have lower growth and higher chances of obstructed labor, one of the major causes of maternal mortality. Zinc supplementation has been found to improve outcomes for pregnancy and child development including reductions in pre-term deliveries, reduced pregnancy-induced hypertension, improved fetal neurobehavioral development, reduced risks of acute diarrhea, dysentery, and impetigo (IZiNCG, 2004). In non-pregnant adults and the elderly, zinc also plays an important role in health, although there is insufficient evidence to quantify it. In adults and the elderly zinc deficiency is associated with degenerative changes from aging, including reduced neurological functioning, decline in immunocompetence, slowed wound healing, and reduced appetite and taste acuity (IZiNCG, 2004). It is increasingly recognized that zinc plays an important role in immunosenescence, the gradual deterioration of the immune systems that occurs with aging (Mocchegiani et al, 2006). There are very few published studies that quantify the contribution to morbidity and mortality of zinc deficiency in any group except children under 5 years of age, despite the recognition of the importance of zinc for this group and the high prevalence of inadequate intakes, and more research is needed in this area (Caulfield et al., 2003). Estimates based on aggregate food supply suggest that 20.5% of the world s population is at risk of inadequate zinc intake, with the highest burden in developing countries (Weuhler et al., 2005). Zinc is often the limiting growth nutrient in diets in populations with a high prevalence of malnutrition (Michaelsen, 2009). Figure 1 below displays the global prevalence of zinc deficiency by region.

25 16 Figure 1: Zinc Deficiency by Region (Source: Wessells, K. R., & Brown, K. H. (2012). Zinc Bioavailability Zinc may be the limiting growth factor in populations with low intakes of animal-source foods because animal-source foods provide the primary source of highly bioavailable zinc in diverse diets (IZiNCG, 2004). Although zinc is also found in high levels in cereals and legumes, the bioavailability of zinc in these foods is low because of antinutrients which inhibit absorption. Antinutrients can be defined as, food constituents that have a negative impact on the solubility or digestibility of required nutrients and thereby reduce the amounts of bioavailable nutrients and available energy in the foods (Michaelsen et al., 2009). Dietary factors such as protein, calcium, vitamin C, and phytic acid influence the proportion of zinc available for absorption. Research shows that phytic acid is the most important inhibitor of zinc absorption, and is found in very high quantities in staple-based diets in developing countries (Sandberg, 1991; IZiNCG, 2004; Michaelsen et al., 2009). Phytic acid is commonly called phytate, which refers to its presence in a salt form bound with other divalent cations.

26 17 Phytate Inhibits Zinc Absorption Phytate is the main storage form of phosphorus in many plant tissues including seeds and grains, and is essential for the germination of the young plant. However, phytate is a strong inhibitor of mineral bioavailability in humans because it bonds strongly with minerals such as zinc, iron, calcium, potassium, magnesium, and manganese. The human digestive tract is unable to degrade the salt compound, and the nutrients pass through the body unabsorbed (IZiNCG 2004; Michaelsen et al., 2009). The adverse effect of phytate on zinc absorption has been observed by numerous in vivo studies in adults and infants (Adams et al., 2002; Davidsson et al., 2004; Perlas and Gibson, 2005). There is no evidence that humans can adapt to chronically higher phytate intake to increase zinc absorption rates. The inhibiting effect of phytate on zinc absorption is linear and dose-dependent, with no upper limit for the inhibitory effect (Navert et al., 1985 in Michaelsen et al., 2009). Any reduction in phytate increases the bioavailability and absorption of zinc (Michaelsen et al., 2009). Therefore, in populations dependent on high-phytate staples, dietary modification through either an increase in zinc or a decrease in phytate will improve the rate of zinc absorption. The inhibitory effect of phytate depends on form of the phosphate molecule. Only the higher inositol phosphates (hexa-phosphate, IP-6 and penta-phosphate, IP-5) inhibit zinc absorption. The higher inositol phosphates can be degraded (hydrolyzed) by the action of the enzyme phytase into lower inositol phosphates (IP-4, etc.) that do not inhibit absorption. Ruminant animals, such as cattle, goats, and sheep are able to digest phytate because their rumen contains microorganisms that produce phytase enzyme. The human digestive tract, however, has insufficient phytase activity to hydrolyze phytate. However there are processing methods that utilize endogenous phytase (already present in the seed) or exogenous phytase (added from an external source), to degrade the phytate in plant foods. Plant

27 18 seeds contain phytase enzyme, which becomes active during germination, releasing the minerals for use by the growing plant. Therefore, the practice of sprouting seeds before consumption can reduce phytate levels. Likewise, lactic acid fermentation can induce phytate hydrolysis through the action of microbial phytase in the bacteria. Because phytate is water-soluble, the process of soaking cracked maize and discarding the water has also been shown to reduce phytate (IZiNCG, 2004). Zinc Physiological Requirements Estimates of zinc physiological requirements have typically been made using a factorial approach, which considers the amount of zinc necessary to offset losses in intestinal and non-intestinal sites (IZiNCG, 2004; Hotz, 2007). The International Zinc Nutrition Consultative Group (IZiNCG) has published revised estimates of mean physiological requirements (MPR) for age and sex groups (IZiNCG, 2004; Hotz, 2007). These revised requirements offer several improvements over previous estimates published by the WHO (1996, 2004) and the Institute of Medicine (2000) and they are considered more appropriate for international use (Gibson, 2012). Despite these improvements, there is still some disagreement about the IZiNCG requirements. For children and women there are some suggestions that the published zinc requirements are too low. For children, the calculations of losses of endogenous zinc are extrapolated from adults, which may result in errors in the estimation. The estimates also do not account for the possibility that zinc absorptive capacity is reduced by chronic intestinal inflammation, known as environmental enteropathy, which is increasingly recognized for its role in child health in developing countries (Korpe and Petri, 2012). There are suggestions that non-pregnant adult women may have average requirements approaching 3 mg/day, over 60% higher than the published requirements (Fairweather-Tait et al., 2012). The zinc requirements for the elderly also requires future research. There is evidence that elderly may have a lower absorptive capacity, which may be offset by a lower rate of zinc loss. More research

28 19 is necessary, however the published IZiNCG currently represent the best figures for the zinc physiological requirements in developing countries, and are used in this study. The effect of using different requirements was also tested. Table 1 shows the mean physiological requirements for zinc by age, sex, and physiological status as per IZiNCG. Table 1: Zinc Mean Physiological Requirements (MPR), IZiNCG 2004: Age, Sex, Physiological Status Zinc Physiological Requirement, (mg/day) 6-11 months years years years years, male years, female 1.98 >18 years, males 2.69 >18 years, female 1.86 Pregnant Women 2.68 Lactating Women 2.98 Bioavailability: Estimates of Zinc Absorption Although several other factors including calcium and protein may influence the proportion of total zinc intake that is available for absorption, phytate is by far the most important predictor of zinc absorption, especially in high-phytate diets (IZINCG, 2004; Miller et al., 2007). The phytate-tozinc molar ratio relates the relative amounts of phytate and zinc in single foods or in diets, and is commonly used to estimate the proportion of absorbable zinc (Gibson, Hotz and Perlas, 2006). The ratio is calculated as follows, where 660 and 65.4 are the molecular weights of phytate and zinc, respectively: mg phytate / 660 mg zinc / 65.4 While animal source foods have zero phytate, unrefined cereals, seeds, nuts, and legumes typically have the highest phytate:zinc molar ratios. Table 2 below shows the total zinc, total phytate, and phytate:zinc molar ratios of several foods including staples in Malawi and Ghana. A ratio above 15

29 20 is considered high enough to substantially limit zinc bioavailability. While a ratio above 15 is considered low availability by the World Health Organization, a ratio between 5-15 is considered moderate availability, and a ratio below 5 is considered high availability (2004). Table 2: Zinc, Phytate, and Phytate:Zinc Molar Ratios of Several Staple foods from Malawi and Ghana (Source: Gibson and Ferguson, 2008; Gibson, 2012) Zinc (mg) Phytate (mg) Phytate: Zinc Molar Ratio Maize flour, 95% extraction Sorghum flour Ground Nuts, boiled Pigeon peas, fresh Kidney beans, fresh Pumpkin leaf Okra Sweet Potato Cassava Gari (dry fermented cassava) Banana Plantain, ripe Chicken thigh, roasted Several models of zinc absorption have been constructed by the World Health Organization (1996, 2004), the Institute of Medicine (2000), and the International Zinc Nutrition Consultative Group (2004), which differ in their assumptions, data sources, and methodologies. The table below displays the predicted zinc absorption as a percentage of total zinc intake for the three different models. Whereas the IOM model provides one estimate of absorption for all groups, the WHO and IZiNCG models predict absorption based on the phytate:zinc molar ratio of the diet. Only the IZiNCG model provides estimates for different age and sex groups, and was previously considered the most appropriate of the three for application in international studies (Gibson, 2012). The IOM model is considered to be extremely limited for application in low-income countries because the data used to derive the estimates were mostly from low-phytate diets in the United States, and severely underestimate the inhibitory effect of phytate (Gibson, 2012).

30 Table 3: Comparison of WHO and IZINCG Estimates of Zinc Absorption Based on Phytate:Zinc Molar Ratios. Source: IZINCG, 2004 WHO (1996, 2004) IOM (2001) IZINCG (2004) Phytate:Zinc Molar Ratio < >15 NA 4-18 >18 Predicted Zinc Absorption 50% 30% 15% 41% 26% men 34% women 31% children 21 18% men 25% women 23 % children However Miller and colleagues have since provided a trivariate mathematical model to predict zinc absorption based on the total zinc and phytate in the diet that offers several improvements over previous models (Miller et al., 2007; Hambidge et al., 2010). This improved model predicts total absorbed zinc from total daily zinc and phytate using a model derived from a biochemical conception of the absorption process. The original model has been validated and parameter estimates refined using subsequently published data (Hambidge et al., 2010). The trivariate model for zinc absorption is: TAZ = 0.5 ( TDZ (1 + TDP 0.68 ) ( TDZ (1 + TDP 0.68 )) TDZ ) Where TAZ is the total daily absorbed zinc, TDZ is total daily dietary zinc, and TDP is total daily dietary phytate. Description of the Model for Zinc Absorption There are several features of this model that merit further explanation. The model is derived from studies of adult men and women and is considered valid for adults of both sexes. The equation represents a biochemical conception of the zinc absorption process based on saturation-response modeling. This saturation model implies that the amount of zinc that can be absorbed in one day approaches a maximum value for a given level of dietary phytate. As the amount of dietary zinc

31 22 increases, the marginal increase in zinc absorbed is lower and lower. The model predicts a maximal absorption of about 6 mg/day of zinc (in a zero-phytate diet), and predicts that the average dietary requirement doubles with approximately every 1000g increase in phytate (Hambidge et al., 2010). In addition, the model does not predict an upper threshold for the effect of phytate to limit zinc absorption. The implications of this are that when dietary zinc and phytate are both high, increasing dietary zinc has negligible improvement; phytate must be reduced to increase the absorbed zinc. The value of the maximum absorbed daily zinc is affected by age, and the value for infants is calculated as less than one-third the value for adults based on relative intestinal length (Hambidge et al., 2010). Nevertheless, the model is not considered valid for very young children because the model is based only on studies of adults, as there were no suitable experimental data for young children. In addition, there is evidence that the role of phytate on zinc absorption in healthy infants is less than previously believed. A new model of data from existing studies found that contrary to expectations, dietary phytate did not have a strong effect on zinc absorption in the limited number of studies of children that currently exist, although there was no immediate explanation (Miller et al., 2015). However phytate reduction may still be important for sick children, as one study found that reducing dietary phytate improved the zinc nutrition of children recovering from tuberculosis, but not that of healthy children (Manary et al., 2000). For children, total zinc, nutrient density, and intestinal health may be more important factors for zinc nutrition than dietary phytate. Ultimately much more empirical evidence is required to establish to mechanisms of zinc absorption in infants and young children. Despite its limitations, this new model of zinc absorption offers a significant improvement over previous models, and suggests that the actual daily requirements for zinc may be as much as double the estimates from the Institute of Medicine at certain high phytate levels (Gibson, 2012). One of

32 23 the strengths of this model is that it allows simulation of the additional absorbed zinc that would result from a reduction in phytate without an increase in dietary zinc intakes. The trivariate model has been used in studies to estimate the impact of zinc biofortification on zinc absorption in Mexico and Bangladesh, and will be used in this study for all groups except infants (Rosado et al., 2009; Arsenault et al., 2010). For infants, total dietary zinc will be compared against the Estimated Average Requirements based on a blanket rate of zinc absorption provided by the World Health Organization (2004). Assessment of Zinc Status of Populations There are several indicators for assessing the risk of zinc deficiency in populations, depending on the resources available and the objectives of the assessment. The International Zinc Nutrition Consultative Group provides three indicators for assessing zinc status: percentage of children less than 5 years old with low height for age (suggestive evidence); percentage of population with low serum zinc concentrations (biochemical evidence); and prevalence of zinc intakes below the estimated average requirement (dietary evidence). While measurement of serum zinc concentrations is the recommended biochemical indictor for assessing the zinc status of populations, data collection is costly and difficult. Suggestive evidence of zinc deficiency, including rates of stunting and estimates of the national food supply, are more readily available but have a number of limitations. Data from National Food Balance Sheets, provided by the Food and Agricultural Organization, have been used to estimate the adequacy of zinc in national food supplies (Wuehler et al., 2005). When combined with stunting rates, this approach can provide an estimate of zinc status, however it still faces numerous limitations. These include possible errors in food balance data, errors in estimated nutrient contents of foods, lack of information on country-specific food processing, and inadequate information about meal size (Wuehler et al., 2005). In addition to these limitations, this approach can only be used at the

33 24 national level and provides no information about regional risk or risk for population subgroups. Although national level estimates suggest that data on subgroups is urgently needed, few attempts have been made to estimate zinc deficiency risk in population subgroups (Gibson, 2012). Analysis of dietary intake data can provide a valid estimate of risk of zinc deficiency and is useful for assessing population sub-groups (IZiNCG, 2004; Gibson et al., 2008; Gibson, 2012). This process involves several steps including sample selection, measuring food intake, calculating zinc and phytate levels from food composition tables, adjusting the distribution of intakes to remove individual variation, and comparison of intakes against published Estimated Average Requirements (Gibson et al., 2008). Zinc deficiency is considered to be of public health concern when the risk of deficiency in a population exceeds 25%. The validity of the dietary intake indicator for assessing zinc deficiency risk in populations has been confirmed by comparing zinc risk predictions based on dietary intake with measurements of serum zinc concentrations (Hotz, 2007; Gibson, 2012). Studies in Malawi, Egypt, Mexico and the United States have found that the predicted prevalence rates using food intake data were very close to the rates using serum zinc (Hotz, 2007). There is less convergence for children between the predictions because the estimates of zinc absorption for children are extrapolated from adults and may be less valid as factors such as infections may limit absorption in children (Hotz, 2007). Despite limitations, the validity of the dietary indicator of zinc deficiency risk has been confirmed by the WHO, UNICEF, and the International Atomic Energy Authority (de Benoist et al., 2007). Dietary indicators for assessment can be especially useful for identifying vulnerable subgroups and evaluating between possible intervention options.

34 Strategies to Improve Zinc Nutrition: Household Food Processing Introduction The International Zinc Nutrition Consultative Group broadly recommends three approaches to improving zinc nutrition: dietary diversification/ modification, supplementation, and fortification (IZiNCG, 2009). Dietary modification includes household processing techniques which can increase the bioavailability of zinc by reducing the levels of phytate in staples such as beans and grains (IZiNCG, 2007). These processing techniques include soaking, germinating, and fermenting and have been used for thousands of years as an essential food security strategy at the household level. Despite their history, acceptance, safety, and cultural relevance, they have been underutilized by development planners and policy makers as potential entry points (Ibnouf, 2012). There is however increased recognition that when poor populations are dependent on plant-based staple foods, they should be processed to reduce the levels of antinutrients (Michaelsen et al., 2009; de Pee and Bloem, 2009). These approaches have the added advantage of being long-term and more sustainable than relying on outside interventions such as fortification or supplementary food assistance. Overview of Benefits of Household Food Processing Approaches Traditional, household-level methods of food processing have been used since before recorded history to increase food storage properties and prevent spoilage, increase nutrients, improve digestibility, remove toxins, improve organoleptic qualities and palatability, and improve consumer desirability (Steinkraus, 1997). Although food processing today has become extremely advanced at an industrial level, many traditional techniques are used around the world including mechanical processing, heating, soaking, germinating, and fermenting. Although some processes can reduce the levels of vitamins and minerals through heat destruction, other methods such as soaking,

35 germinating, and fermenting can both increase nutrient levels and increase the bioavailability of existing nutrients through reduction of phytate. 26 Soaking Soaking is widely used around the world for legumes, grains, and flours, and typically occurs for a period of several hours up to several days. During soaking, phytate diffuses into the soaking water, which is subsequently discarded, thus removing the phytate. Even short periods of soaking can reduce the phytate content of staple foods, although the extent of reduction depends on the time lengths and conditions of soaking and the food product. Soaking in contaminated water may pose health risks if it is not adequately boiled to kill pathogens before consumption. Studies have found that the bioavailability of proteins is also increased up to 50% after soaking for some grains (Mensah and Thompkins, 2003). Studies have found reduced phytate and increased acidity (reduced ph levels) from soaking. Hotz and Gibson (2001) thoroughly reviewed the potential of traditional household processing techniques to reduce the phytate content of cereals and legumes. Soaking of cereal and legumes reduced the phytate content of maize flour by 50%, and of legumes by 20%. A 50% reduction in phytate through soaking was achieved with Malawian maize in both laboratory and practical field settings (Hotz, Gibson, and Temple, 2001). Other studies have shown variability in amount of phytate reduced from soaking, and the level of reduction was lower in some cases. The degree of phytate reduction appears to be based on the morphological location of phytate in the seed and whether it is diffused into the soaking water. There may also be loss of water-soluble nutrients that occurs when the soaking water is discarded (Hotz and Gibson, 2001).

36 27 Germination and Malting Cereal crops of the poaceae of the monocotyledonic branch of the plant kingdom constitute some of the most important human food crops globally, and include wheat, rice, maize, barley, and sorghum, as well as crops of regional importance such as rye, millet, and oats. Many around the world are dependent on cereals as a source of energy, protein, fiber, vitamins and minerals. Legumes and grains which form the seed of a plant can be germinated through a process of soaking allowing the seed to sprout. After germinating, cereals are either consumed as sprouts or dried and milled. Hübner and Arendt (2013) conducted a review of germination as a way to improve the nutritional value of cereal grains. Germination reduces the phytate content and increases the bioavailability of certain nutrients in staple foods. In Malawi, germination of maize reduced phytate levels by 46%, increasing the extractability of calcium and iron 100% (Hotz and Gibson, 2001). In one study of brown rice, germination caused a phytate decrease of 60% (Liang at al., 2008), while a study of sorghum reported an 87% decrease in phytate after 4 days (Mahgoub and Elhag, 1998). In addition to phytate degradation, germination reduces viscosity, promotes growth of probiotic bacteria, and increases vitamin levels. As complex starches are broken down into simple sugars, cereal porridge becomes thinner and can be consumed by infants without dilution. Mensah and Thompkins (2003) report that this thinning effect is the most important nutritional effect of germination, because thinning by dilution with water increases the risk of contamination and decrease the nutrient density. Similarly, the addition of enzyme-rich flour (from germinated grains) to non-germinated flour can increase energy intake from weaning foods (Michaelsen et al. 2009; Mensah and Thompkins, 2003). Germination also increases the amount of fermentable sugars available to desirable probiotic bacteria which promote intestinal health. Studies have found germination as an effective way to

37 28 increase folate, an important nutrient for preventing neural tube defects, in oat, barley, wheat, and rye (Hübner and Arendt, 2013). Other B Vitamins have also been found to increase a results of the germination processing including riboflavin, pantothenic acid, and pyridoxine (Briggs, 2001 in Hübner and Arendt, 2013). Despite these benefits, there are risks associated with the germination procedure at the household level. Germination requires soaking which may promote growth of bacteria and fungi, and the process is time- and space-consuming. Malting or germination may have cultural associations with the alcohol brewing process, and may be unacceptable in societies where alcohol is prohibited. Fermenting Fermentation has been practiced as a method of indigenous food processing since before recorded history, and fermented foods today make up a large proportion of diets in both modern and traditional food systems (Steinkraus, 1995). Fermentation can be generally described as desirable changes brought about by microorganisms. More technically, fermented foods are food substrates invaded by edible microorganisms whose enzymes (in particular amylase, protease, and lipase) hydrolyze the polysaccharides, proteins, and lipids to form non-toxic products and produce flavors, aromas, and textures that are pleasant and attractive to the consumer (Steinkraus, 1995). Foods can be fermented by the microorganisms that already exist in the foods (spontaneous fermentation) or by deliberately adding a starter culture of microorganisms to the food substrate. In many cultures, although the science behind fermentation is not understood, practices have evolved to ensure that an adequate amount of microorganisms are present in each batch. Such practices may include reusing the same vessels in which the yeast or bacteria has colonized, using a particular instrument to stir the mixture which insures proper inoculation, or adding some of a previous batch of the fermented food product ( back-slopping ). In some cases, leaving a

38 fermenting mixture exposed to the open air ensures that desirable microorganisms from the local environment colonize the mixture and initiate fermentation. 29 The biological processes that occur during fermentation are complex and vary based on the food substrate, microorganisms, and conditions of the fermentation. They may include the breaking down of proteins into peptides or amino acids, breaking starches into simple sugars, the conversion of sugars into ethanol (alcohol) and carbon dioxide, and the production of phytase enzyme. Fermentation is one of the most effective methods for preserving foods. The lower ph resulting from bacterial production of organic acids prevents the growth of spoilage organisms, increasing the shelf life and safety of a food product (such as preventing milk spoilage or preserving cheeses for the lean season) (Michaelsen et al., 2009). In addition to enhancing the sensory and organoleptic properties of foods, fermentation increases the bioavailability of minerals such as zinc, lowers the ph to prevent growth of spoilage organisms, increases the levels of probiotics and B Vitamins, and reduces anti-nutritive components such as cyanide in cassava. Fermentation can prevent contamination of infant weaning foods, a major contributor to diarrhea, one of the principal causes of under-five mortality (Motarjemi et al., 1993). Fermentation may also be used to improve weaning foods by increasing viscosity and energy density, and may be combined with other processing methods to increase benefits. A tremendous range of fermented foods has been recorded around the world and throughout history. Commonly fermented foods include grains, roots, seeds, milks, fruits, fish and vegetables. Table 4 below lists a number fermented foods selected to show the diversity of fermented foods in Sub- Saharan Africa and in several other regions.

39 Table 4: Selected Fermented Foods (Sources: Steinkrauss in Mensah and Thompkins; Aworh 2008; Ibnouf 2012 ) Fermented Food Geography Substrate Usage Gari, Fufu, Chikwangue, kwanga, lafun West Africa Cassava Doughy, Paste, staple seasoning; soft condiment Koko, akasa, kenkey, Ogi (uji), akamu, banku Ghana, West Africa Maize Porridge, dumplings kito East Africa Maize Porridge Poto-poto Africa, Congo Maize Weaning gruel Thobwa Malawi Maize Non-alcoholic fermented beverage Ntoba mbodi Congo Cassava leaves Condiment Bikedi Congo Cassava Grated plant Dawadawa (iru, kpalugu) West Africa Locust bean Condiment Ugba Nigeria Oil bean seeds Delicacy and flavoring Warankasi Nigeria Milk Soft cheese Kpaye (okpeye) Nigeria Seed of P. Condiment Africana Ogiri Nigeria Melon/ pumpkin Condiment seed Nasha, Kisra Sudan Sorghum Porridge/ bread Roob, kush-kush, mish Sudan Milk Yoghurt, soft cheeses Oshikundu, Mahewu, pombe, burukutu, pito, otika, shekete, kpaye, dolo, bili-bili, Merissa, agadagidi, busa Kawal Sudan Cassia plant Meat substitute leaves Njera Ethiopia Maize or teff Pancake/ flatbread Obusera East Africa Millet Porridge Bogobe Botswana Sorghum Porridge Africa Sorghum, millet, maize, cassava, plantain, dates, spices 30 Traditional Cloudy Beer Dhokla, dhosa, idli India Gram, wheat, rice Spongy pancake bread Radbi India Maize, milk Buttermilk, cheese Fish sauce (nuocmam, patis, mampla) Southeast Asia Fish Condiment, seasoning agent Soy Sauce Japan, China, Soybeans, wheat Condiment others Natto Japan Soybeans Meat substitute Oncom Indonesia Peanut Cake, meat substitute Tempeh Indonesia, Asia Indonesia, SE Meat substitute Asia Pozol Mexico Maize Spongy dough Chicha South America Maize, spices Alcoholic drink

40 31 Household Processing Methods Increase Zinc Bioavailability Phytate inhibits the absorption of zinc, iron, calcium, and other minerals. When phytate is degraded through hydrolysis by the enzyme phytase, those minerals become available for human absorption. Processing promotes the hydrolysis of phytate through the production of phytase by microorganisms and the activation of endogenous phytase in the grain, as well as through passive diffusion of water-soluble phytate into soaking water. The activity of endogenous phytase enzyme increases as a result of the lower ph from the organic acids produced during fermentation. These citric, lactic, and acetic acids also form soluble ligands with the minerals, which prevents them from forming insoluble complexes with phytate and this makes them available for absorption (Sandstrom, 1997 in Gibson, Hotz, and Perlas, 2006). The potential of fermentation to improve zinc bioavailability depends on the degree to which the phytate is reduced. Studies have found a wide range for the degree of phytate reduction depending on the food substrate and prevailing conditions of fermentation. A comparison of the level of phytate reduction for various substrates in selected studies is shown in Table 5. Table 5: Summary Table of Studies of Processing on Maximum Percent of Phytate Reduction Substrate Maximum Percent of Phytate Reduction Literature Source (Method) Maize (Malawi) 51% (soaking) Hotz, Gibson, and Temple, 2001 Maize (Malawi) 12% (fermentation only) Hotz and Gibson, % (combination of methods) Maize (Nigeria) 58% Marfo et al., 1990 Maize porridge 72% (white maize) Adewusi et al., 1991 (oji) (Nigeria) 88% (yellow maize) Pea Cultivars 37% (soaking, de-hulling, germination) Duhan et al., 1999 Pearl Millet 50% (malting) Archana et al., % (blanching) Cereals/ 100% (soaking, germination, Sandberg, 1991 vegetables fermentation) Cereal gruel 50% (germination) Svanberg, et al., % (fermentation) African Yam Bean 30% (fermentation) Ene-Obong et al., 1996 Sorghum 65% (fermentation) Makokha et al., % (malting) Finger Millet 72% (fermentation) Makokha et al., 2002

41 32 A simple method of pounding Malawian white maize, soaking, then decanting excess water was found to reduce phytate by 51% (Hotz, Gibson, and Temple, 2001). An educational pilot program was conducted in rural Malawi and participant samples were tested to contain an average of 48% of the phytate of unprocessed maize, thus confirming the efficacy of phytate reduction using this method in a practical setting. Another study of Malawian white maize that was soaked, germinated, and fermented under laboratory conditions examined the extent of phytate reduction. Lactic acid bacteria fermentation reduced phytate to 88% of controls, whereas combination of fermentation with other methods reduced phytate to 54% of controls, which though significant, was a smaller reduction than in other studies (Hotz and Gibson, 2001). These differences may have resulted from method of phytate measurement, the characteristics of the lactic acid bacteria responsible for fermentation, and other environmental conditions. Processing maize into traditional ogi porridge through fermentation reduced phytate 72% and 88% in white and yellow maize, respectively (Adewusi et al., 1991). Marfo and colleagues measured the phytate reduction from traditional fermentations of white and yellow maize, which showed a 58% and 55% reduction in phytate, respectively. Reductions were even greater for other foods, including an 80% reduction for rice, and a 98% phytate reduction in cocoyam. Additional phytate reduction was found when maize was processed into the final products eko (87% total phytate reduction), koko porridge (86%), and kenkey (60%) (Marfo et al, 1990). The authors concluded that local methods of food processing are effective in reducing the negative effect of phytate. Pea cultivars subjected to soaking, dehulling, cooking, and sprouting all showed a reduction in phytate, with 48-hour germination having the greatest reduction (Bishnoi et al., 1994). High-yield pea cultivars developed by ICRISAT had a 4-37% reduction in phytate and increased solubility of iron, calcium, and phosphorous when subjected to soaking, dehulling, cooking, and germination (Duhan et al., 1999). Pearl millet grains were malted and blanched to reduce both phytate and antinutritional polyphenols. Blanching reduced phytates and polyphenols 38% and 28%, while

42 malting reduced them 50% and 48%, respectively. Malting for 72 hours was the most effective technique in the study for reducing antinutrients (Archana et al., 1999). 33 A study on the combined effect of soaking, germinating, and fermenting on phytate hydrolysis found that the combined processing methods were able to completely reduce phytate in cereals under optimal conditions, thus greatly increasing the bioavailability of zinc, iron, and calcium (Sandberg, 1991). Another study compared the effects of germination, natural fermentation, and addition of exogenous phytase enzyme on phytate reduction in cereal grains. Gruel made from germinated flour had a 50% reduction in phytate, while soaking followed by fermentation reduced phytate 90%. The addition of exogenous phytase enzyme reduced the phytate level to just 3% of controls (Svanberg et al., 1993). African yam bean, an under-utilized legume from Nigeria was subjected to soaking, dehulling, natural fermentation, and heat treatment (Ene-Obong and Obizoba, 1996). While soaking reduced cooking time and phytate levels, dehulling led to the loss of important nutrients in the hull. Fermentation, however reduced phytate levels by 20-30%. In another study, sorghum and finger millet from Kenya was malted and fermented, and the phytate levels were measured across several time intervals (Makokha et al., 2002). Fermentation decreased phytate in sorghum by 39% after 72 hours and 65% after 96 hours. The corresponding reductions for finger millet were 54% and 72%. Malting reduced phytate 24-45% over the same time intervals. The study concluded that fermentation was more effective than malting in increasing the availability of iron, manganese, and calcium. A study of traditionally fermented breakfast foods of India demonstrates that the initial level of phytate in the food substrate is an important determinant of the success of processing to improve mineral availability (Hemalatha et al., 2007). Fermentation of dhokla, which has high initial levels of phytate from chickpeas, did not substantially improve iron or zinc availability. In contrast, the fermentation of idli and dhosa, traditional foods made from rice and lentils, had an

43 enormous increase in bioavailability of iron (up to 277%), and an increase in zinc availability of 50-71% Strategies to Improve Zinc Nutrition: Alternatives to Household Processing In addition to dietary modification, the International Zinc Nutrition Consultative Group recommends supplementation, fortification, and biofortification as possible strategies to improve zinc nutrition (Brown and Hess, 2009). Although there is evidence that targeted zinc supplementation can improve health outcomes, supplementary and therapeutic feeding approaches may be limited in their ability to address widespread zinc deficiency. While effective for treating severe acute malnutrition in subgroups, they are less feasible for addressing widespread zinc deficiency at the population level. Fortification and supplementation programs may be limited to urban areas and rural areas with good infrastructure. In addition, supplements may displace local foods and breastmilk. In a review of the dietary advice given to caregivers by major agencies and donors, the authors found that there is a greater emphasis on providing food supplements than on improving local diets (Ashworth and Ferguson, 2009). Biofortification offers still another approach and holds great potential for increasing the dietary zinc intakes of large population subgroups. Zinc Supplementation Trials indicate that zinc supplementation reduces the incidence of pneumonia and diarrhea in children, and increases linear growth in previously stunted children (Brown and Hess, 2009). Zinc supplementation has been found to reduce incidence of diarrhea by 27% among young children, lower respiratory tract infections by 15%, and may reduce malaria incidence (Brown and Hess, 2009). Evidence also suggests that maternal zinc supplementation may reduce the rates of preterm births and postnatal infections, and may increase infant growth (Brown and Hess, 2009). However zinc supplementation depends on government support and stable distribution infrastructure (Meenakshi et al., 2010). In addition, it lacks sustainability in that as soon as the

44 program is discontinued, recipients no longer have a reliable zinc source. Other challenges to zinc supplementation include non-compliance, as people may forget to take them (Suarez, 2011). 35 Ready-to-Use-Therapeutic Foods (RUTFs) Community management of acute malnutrition (CMAM) is used when there is a high prevalence of severe wasting, and may be effective to address zinc deficiency. It is used for children suffering moderate and severe acute malnutrition, as well as pregnant and lactating mothers. It typically utilizes a case-finding and triage approach in which most children are treated in their homes while those with medical complications are referred for in-patient treatment, and uses Ready-to-Use- Therapeutic Foods (RUTFs). RUTFs used in Community Management of Acute Malnutrition are an important contributor to child survival among populations with limited access to quality foods and high prevalence of child malnutrition including severe wasting (De Pee and Bloem, 2009). Studies have found ready-to-use therapeutic foods (RUTFs) are very effective in treating children with severe wasting, in part because of their very high energy density (Michaelsen et al., 2009). However there is question as to whether they are appropriate for use outside of cases of severe wasting, such as zinc deficiency. The sustainability of the CMAM model of supplementary feeding is limited by reliance on outside support. The use of RUTFs for treatment of moderate acute malnutrition (MAM) probably provides excess energy, and it is not realistic for reaching the large number of children with MAM due to high cost of the foods and low production capacity (De Pee and Bloem, 2009). Because it is a targeted intervention, it does not reach household members who are not direct recipients. A review of the evidence of home-based use of RUTF found that there is insufficient evidence to recommend the use of RUTF over a standard home diet of such as flour porridge (Schoonees et al., 2013).

45 36 Fortified Blends Fortified food blends typically contain about 25% soybeans and 75% wheat or corn, and may contain sugar, oil, or legumes. They can be prepared into a porridge by parents at home to feed children. Although these blends are reasonably nutritious, relatively low-cost, and can be produced anywhere, they have limitations. The main buyers of these blends are USAID, WFP, and UNICEF, and reliance on external donors can create dependency and undermine long-term sustainability (de Pee and Bloem, 2009; Lazzerini et al., 2013). In addition, they may be limited for addressing zinc deficiency because of the high level of phytate which reduces micronutrient bioavailability. Cornsoy blends procured by UNICEF and WFP made from whole soybeans and corn have high levels of the phytate (de Pee and Bloem, 2009). Weanimix, a blend of cereals, legumes, and nuts introduced in Ghana in 1987 by UNICEF, was found to have a phytate:zinc molar ratio of 17, above the WHO threshold for low zinc bioavailability (Mensah and Thompkins, 2003). In a thorough review of options for addressing moderate malnutrition, the authors conclude that, for feeding young or malnourished children, fortified blended foods need to be improved or replaced, and that data are urgently required to compare the impact of new or modified commodities with that of current fortified blended foods and of RUTF developed for treating severe acute malnutrition (de Pee and Bloem, 2009). Shortcomings of Supplementation and Fortification for Addressing Zinc Deficiency Approaches that focus only on increasing the dietary protein through the use of cereal/legume blends has ignored the problems associated with micronutrient and mineral availability. Food aid products are often high in phytate which reduces the bioavailability of zinc and iron (WHO, 1998; Gibson et al., 1998; Gibson et al., 2010; Gibbs et al., 2011; Roos et al., 2013). A study comparing 26 indigenous home-prepared and 27 commercially-processed plant-based complementary foods from low-income countries found consistently low levels of nutrient

46 37 bioavailability (Gibson et al., 2010). All but two of the commercially-distributed processed foods had excessive phytate above the target ratios. Another study of 57 processed complementary foods from Africa and Asia marked specifically for infants found that many had low in nutrient levels or low nutrient bioavailability due to phytate (Gibbs et al., 2011). Only 2% of the foods studied met the zinc requirements of breastfed infants, despite fortification. A third study screened the anti-nutrient levels of 36 RUTF products used in food aid programs by international agencies, local blended foods, and fortified instant porridges for infant feeding (Roos et al., 2013). Findings confirmed that despite fortification, a number of products did not meet the recommended mineral levels and that phytate levels were consistently high. Eleven of 36 products exceeded the phyate:zinc ratio of 15 (indicating low bioavailability based on WHO criteria), while another 15 products had a phyate:zinc ratio between 5 and 15 (indicating only moderate bioavailability). These findings support the notion that there is an urgent need to improve the nutritional quality of plant-based foods in developing countries, especially those used for feeding infants and young children (Gibson, Hotz, and Perlas 2006). Biofortification Biofortification seeks to improve the nutritional content of staple food crops through conventional plant breeding or through the use of transgenic techniques (Bouis et al., 2011; Saltzman et al., 2013). This approach takes advantage of the consistently high consumption of certain staple crops by poor households in developing countries, as well as the high intake of these staples by all household members. Although biofortified crops cannot deliver levels of micronutrients as high as supplements, they offer a number of advantages. In contrast to fortification and supplementation programs, biofortification is designed to reach decentralized rural populations. Unlike supplementation and

47 38 fortification programs which require continued financial support, biofortification only requires a one-time investment to develop new plant breeds, which farmers continue to grow, providing a sustainable multiplier effect. Biofortification also faces limitations and challenges. For those with severe nutrient deficiencies or special needs such as pregnancy, supplementation may still be necessary. Because biofortification is still an emerging technology, it will continue to take much time before new varieties are developed, disseminated, and evaluated. HarvestPlus identifies three conditions for biofortified foods to be successful: breeding must yield high nutrient density; efficacy in increasing nutrient status must be demonstrated when processed, cooked and consumed; and finally, biofortified crops must be adopted by farmers and consumed by those with malnutrition (Bouis et al., 2011). If new varieties vary from traditional ones in appearance, taste, or texture, this may pose a formidable barrier to their successful adoption. Zinc-biofortified wheat and beans have successfully been developed and implemented in India, China, Brazil, Rwanda, Nigeria, and Democratic Republic of the Congo (Saltzman et al., 2013). Although maize has been successfully Biofortified with Provitamin A Carotenoids, maize biofortified with zinc is a very recent development. An experimental high kernel-zinc maize variety is being developed by the International Maize and Wheat Improvement Center (CIMMYT) (Chomba et al., 2015). This new variety has zinc levels of 3.4 mg/100 grams. Evidence suggests that zinc biofortification is effective to increase absorbed zinc in humans. A trial comparing the experimental high-zinc maize variety from CIMMYT to a traditional variety in Zambia found that absorbed zinc was significantly greater among a randomly-selected group of children consuming the biofortified variety over the traditional variety (Chomba et al., 2015). This occurred despite the fact that the phytate level was also higher in the biofortified variety; nevertheless, the phytate:zinc molar ratio was slightly lower (38:1 vs. 34:1), thus enhancing

48 39 absorption. This is consistent with other studies using biofortified wheat. Women in Mexico showed significantly greater total absorbed zinc from biofortified wheat compared to a nonfortified traditional variety (Rosado et al., 2009). Phytate reduction cannot typically be achieved through biofortification because the reduced plant phosphorous results in lower crop yields (Bregitzer et al., 2006). However there are currently trials for genetically modified sorghum with phytate levels reduced (35-80%), expected for release in 2018 (Saltzman et al., 2013). The success of biofortified crops will depend on their coverage or adoption rates by target populations, and this will require assessment in large-scale implementation trials. It is estimated that adopted rates will range from 30-60% in Asia (rice and wheat), and from 20-40% in Africa (maize) (Hotz, 2009). An important consideration for biofortification is the proportion of a staple crop grown imported for consumption, and therefore not biofortified in country. Simulations have been used to estimate the effect of biofortified crops on population prevalence of zinc deficiency. Denova-Gutiérrez et al. (2008) simulated the impact of biofortification of maize, wheat, rice, and beans with iron and zinc on Mexican women and children, using dietary intake data from Mexican National Nutritional Survey. The analysis found that the simulated biofortification of maize with zinc resulted in a significant decrease in the prevalence of inadequate zinc intake. In rural areas, the reduction in prevalence of inadequate intakes was 46% for preschool children and 56% reduction for women. This study confirmed that maize, the primary staple of the rural population, was the most important fortification vehicle. Arsenault et al., (2010) used a similar approach to estimate the impact on zinc nutrition of women and children of rice biofortified with zinc in Bangladesh. This study simulated the adoption of biofortification at 15%, 35% and 70% of the population, and found that biofortification could substantial ameliorate the currently high levels of zinc deficiency.

49 40 Alternatives to Household Processing: Conclusion Although they may be effective under certain circumstances, each of the approaches for improving zinc nutrition reviewed here faces certain limitations. Supplementary approaches, including both direct preventative supplementation and therapeutic feeding using RUTFs are limited in that they rely on continued outside support and only reach targeted recipients, rather than entire households. Zinc fortification, typically of flour blends, requires a stable food processing infrastructure and distribution network, and may not be effective in reaching rural areas. In addition, studies have found that zinc bioavailability may be limited because phytate levels in many of these products are high. Biofortification represents a promising alternative approach to sustainably increase the dietary zinc intakes of rural populations. However requirements of large up front time and financial costs for research and dissemination limit the impact in the short term. Household level dietary modification through processing techniques such as soaking, germinating, and fermenting represent a sustainable, low-cost, and empowering approach to improve household level zinc nutrition through education and behavior change. Because biofortification offers the most attractive alternative to dietary modification, it will serve as the basis of comparison for this study. 2.6 Malawi: Country Overview and Study Population Background Country Profile Malawi is a landlocked country in Southeast Africa (Figure 2). It is one of the poorest and least developed countries in Sub-Saharan Africa, ranking number 174 out of a 187 in the 2014 Human Development Report with no movement in the preceding five years (WFP, 2015). Malawi s population is approximately 16.7 million with an average family size of 5, and it is growing rapidly at a rate of 3% per year. HIV, which affects 11% of the population, and limited access to health care are contributors to the high maternal and child mortality rates (FAO, 2008). The population

50 41 literacy rate was 71.3% in 2013, while total female literacy was 63.9%, and literacy of girls ages only 50% (WFP, 2015). Over 55% of Malawians live below the $1 a day poverty line (Mkwambisi, 2011). Malawi consists of three distinct regions: Northern (composed of six districts), Central (nine districts), and Southern (thirteen districts). The dominant geographical feature traversing the country from north to south is a deep trough forming part of the Great Rift Valley, in which Lake Malawi is located (Figure 2). In the south, The Shire River runs from Lake Malawi to the Zambezi River, while the central region is mountainous. The Southern region is densely populated and contains the biggest urban population and commercial sector. In the highlands of the southern region, soil conditions are favorable and a greater proportion of the population is food selfsufficient. However the Southern region also more vulnerable to flooding and drought than the other regions. The Northern Region is generally rural and isolated. The central region s great plain falls in between, but has been prone to drought and increasing population pressures on livelihoods including low pay rates and high prices for inputs for maize (Malawi Vulnerability Assessment Committee, 2005). Approximately 18.25% of household are located in urban areas. The urban growth rate was 6.3% in 2012, and a quarter of the urban population is estimated to be living in poverty (Mkwambisi, 2011). The largest urban area, Blantyre City, is located in the center of the Southern Region and is the commercial and industrial center of the country. The capital city, Lilongwe, is located in the fertile central region of the country, on a plain 1100 meters above sea level. The two other primary urban areas are Mzuzu City, in the Northern Region, and Zomba, in the South. As of June 2015 there were over 23,000 refugees in Malawi, mostly from the Great Lakes Region, Ethiopia and Somalia (WFP, 2015).

51 42 Figure 2: Malawi Location in Africa and Districts and Regions of Malawi Institutional Context and National Nutrition Priorities in Malawi Micronutrient malnutrition is a national priority in Malawi as evidenced by a number of institutional and policy structures in the country. The office of the president in Malawi has a Secretary for Nutrition, HIV, and AIDS, who leads a Department of the same name. The Department of Nutrition, HIV, and AIDS was established in 2004 and is mandated to lead the integration of nutrition concepts into other departments including Ministries of Health, Agriculture, Gender, Information, Civic Education, and Local Government.

52 43 The Secretary for Nutrition, HIV, and AIDS also chairs the National Nutrition Committee which provides technical guidance on implementing the Nutrition Security Policy (2007) and National Nutrition Policy and Strategic Plan ( ), both of which are being reviewed and revised as of the latest available updates. The national nutrition strategic objectives align with the Scaling Up Nutrition movement (SUN), which Malawi was one of the first countries to launch in Malawi s Cost of Hunger report by the Minister of Finance, Economic Planning, and Development is used as a tool for resource mobilization and recognizes micronutrient malnutrition as a priority (WFP, 2015). Five priority outcomes recognized in the National Nutrition Policy and Strategic Plan are: improved maternal nutrition and care; improved infant and young child feeding practices; improved intake of essential micronutrients; prevention and treatment of common infectious diseases; and improved management of acute malnutrition (USAID, n.d.). The National Nutrition Guidelines for Malawi specifically promote processes such as germinating and fermenting to improve the nutritional value of foods, and Malawi has also prioritized community-based action to reduce stunting through behavior change and education (Malawi Ministry of Health, 2007). The World Bank and the Scaling Up Nutrition initiative estimate that Malawi loses over $600 million USD per year in GDP to micronutrient deficiencies, and that scaling up core interventions would cost less than $9 million per year (World Bank, n.d.). Malawi currently invests 13% of its national budget to address agriculture and food security issues (USAID, n.d.). The institutional structure for promoting improved nutrition is decentralized, with District Nutrition Coordination Committees, Village Development Committees, and Community Leaders for Action on Nutrition at the local levels. The Government of Malawi is supported by institution donors such

53 as USAID and Irish Aid, the United Nations, private sector partners, and The Civil Society Alliance in Malawi (CSONA), which is chaired by the NGO Concern Worldwide. 44 Economy, Agriculture, and Food Security in Malawi The economy of Malawi is based primarily on agriculture, which contributes 35-50% of the Gross Domestic Product (FAO, 2008). Over 80% of the population are smallholder, rain-fed subsistence farmers with less than 0.23 hectares of land, below the average of 0.40 ha for Sub-Saharan Africa (FAO, 2008). The livestock sector is underdeveloped. The economy of Malawi faces shocks such as high inflation and currency devaluation, and ranked the 13 th worst-performing economy in the Global Competitiveness Report. Corruption is a large problem in Malawi. In the Cashgate Scandal, an estimated USD 60 million (1-2% of GDP) of government funds were stolen by public officials, negatively effecting social service delivery (WFP, 2015). The primary agricultural crop grown throughout the country is maize, which constituted over 90% of total cereal output in the 2015 harvest (2,877,000 tonnes), while other crops grown regionally include rice (120,000 tonnes), sorghum (85,000 tonnes), pulses, groundnuts, and cassava. Tobacco, constitutes the largest export cash crop followed by wheat, cotton, tea, sugar, and coffee (FAO, 2015). Despite bumper crops in recent years, the 2015 maize harvest was 27% lower than 2014 and 22% lower than the average yield. While the earlier success was due to favorable conditions and government input subsidies, the most recent period was affected by an extended dry period and extensive flooding in the south which destroyed cropland, livestock, and infrastructure. Accordingly, food prices have been climbing since 2014 reaching highs in July 2015, including during the harvest season when they are typically the lowest (FAO, 2015). Food insecurity in Malawi is primarily caused by lack of access to food due to high prices, rather than lack of food availability. Households further from population centers have lower dietary

54 45 diversity due to lack of market access, are insufficiently integrated local markets, and more of their consumption comes from home production (Luckett et al., 2015). Maize is planted in September through November and grown during December through February, and therefore this growing season is when most of the farming takes place, and food prices are highest and intakes are lowest (Siyame et al., 2013). Adverse shocks during this period, such as droughts, have drastic consequences. Nutrition, Health, and the Causes of Morbidity in Malawi Malawi has very high rates of protein energy malnutrition and micronutrient malnutrition, and is extremely vulnerable to drought-induced famine. The prevalence of stunting is estimated at 48.3%, and micronutrient deficiencies are common for men, women, and children (IZiNCG, 2004; Tinna et al., 2013). Fifty-nine percent of preschool children and 38% of all school children have Vitamin A deficiency. Anemia affects three quarters of children under 5 and nearly half of adult women. Based on national food balance sheet data, 34% of the population is estimated to be at risk of inadequate zinc intake at the national level, and local studies have found zinc deficiency rates as high at 90% (IZiNCG, 2004; Siyame et al., 2013). The Food and Agriculture Organization has stressed the critical need for long term, food based strategies in addition to supplementation and fortification, to address the nutritional crisis in Malawi (FAO, 2008). According to the World Health Organization, the primary causes of infant mortality in Malawi include pneumonia (23%), underweight (22%), diarrheal disease (18%), and malaria (14%). After the first year of life, major causes of mortality include infectious diseases. The primary causes of maternal mortality include hemorrhage, infection, unsafe abortion, eclampsia, and obstructed labor, while indirect causes include malaria, anemia, AIDS, and tuberculosis (WHO, n.d.). Although WHO notes that the maternal mortality ratio declined as the government expanded access to health services, access to health care is still very limited especially in rural areas.

55 46 Improved household processing techniques represent an opportunity to directly address the major causes of maternal and child morbidity that aligns with national priorities of Malawi. Zinc deficiency is directly related to the risk of mortality from diarrhea, pneumonia, and malaria as well as other infectious diseases, which are the primary causes of infant mortality, as noted above (Black et al., 2003). For maternal health outcomes, improved zinc nutrition can reduce morbidity and mortality from prolonged labor, placental abruption, premature rupture of membranes, preeclampsia, hemorrhage, and postpartum malnutrition and infection (IZiNCG, 2004). The low bioavailability of zinc in the typical Malawian diet is one of the major causes of zinc deficiency, stunting and impaired immuno-competence (FAO, 2010). Zinc deficiency may also be a factor limiting pregnancy outcomes for Malawian women, according to one study (Huddle et al., 1998). Maize provided over two thirds of the energy, protein, and zinc for the women, while animal foods (mainly fish) provided less than 5% of energy. Over 60% of the women had a phytate to zinc molar ratio greater than 15, indicating low zinc bioavailability. The authors concluded that because increasing the amount of zinc in the diet was not economically feasible for this population, [A]ny strategy to alleviate dietary zinc inadequacies must emphasize ways of improving the bioavailability of zinc in existing Malawian diets rather than their zinc content. The promotion of improved household-level food processing techniques is appropriate given the current state of health care access in Malawi. This intervention represents a decentralized approach that does not depend on access to health centers or stable infrastructure such as supply chains, as do zinc supplementation and flour fortification. This approach is sustainable and empowering as it builds upon local knowledge using education and behavior change approaches. Dietary Patterns, Food Choice, and Food Preparation Practices in Malawi Maize is the staple crop of Malawian diets, contributing over 60% of total energy and zinc and half of the total protein to the diet (Tinna et al, 2013). Despite Malawi s high agricultural potential, diets

56 47 lack diversity and are poor in micronutrient-dense foods such as animal foods, fruits, and vegetables. Lake Malawi provides an important source of iron- and zinc-rich fish, however fish caught are typically sold in local markets rather than consumed by rural households (FAO, 2010). Although breastfeeding is universal, exclusive breastfeeding to six months is not widespread and child weaning foods are insufficient. Weaning foods typically consist of a thin porridge of refined maize, which is high in anti-nutrients and low in energy and micronutrient density (FAO, 2010). The primary maize staple is a stiff porridge called nsima, which is typically consumed with relish from green leaves, legumes, and sometimes fish if available. Research by the International Food Policy Research Institute confirms the centrality of nsima in the national Malawian diet, a preference which is deeply rooted in tradition and upbringing (IFPRI, 2015). Meals without nsima are often not properly considered food and food security is often defined in terms of whether or not there is enough maize in household stores to meet yearly food needs. As a coping mechanism during scarcity, thin maize porridge may be prepared instead of nsima, effectively by increasing the water-to-maize ratio which lowers the nutrient density. Because it is maize based, however, nsima has high levels of dietary fiber and phytic acid, inhibitors of zinc and iron absorption. Soaking, germination, and fermentation of maize is not widely practiced in the preparation of maize based staples in Malawi (Ferguson et al., 1993; Hotz and Gibson, 2001). In one study of child feeding practices, only 1% of mothers reported ever using fermented maize porridges. Only 10%, 4%, and 2% reported ever using soaked maize kernels, soaked maize flour, or germinated flour for porridge preparations (Hotz and Gibson, 2001). Although not commonly used for staple food preparation, processing methods such as fermentation are not completely foreign in Malawi. A lactic-acid fermented, non-alcoholic maize-based beverage called thobwa is commonly produced in Malawi for consumption at social events and as an energy drink while doing manual labor (Tinna et al., 2013; Kitabatake et al, 2003). Local knowledge and acceptance of this process, which involves control of temperature and time to allow acidification,

57 48 could be transferred to the adoption of improved food preparation techniques. However, preferences for traditional nsima production methods would need to be addressed through nutrition education and behavior change communication programs. Review of Empirical Evidence to Support Behavior Change Interventions for Nutrition Evidence suggests that behavior change communication and nutrition education can effectively and sustainably improve nutrition outcomes. In a detailed literature review, Hess and Brown concluded that nutrition education focused on dietary diversification and modification can have an impact on behavior change and ultimately nutritional status (Hess and Brown, 2009). A study in in Peru described by the reviewers as the most convincing evidence for the impact of behavior change on nutritional status, specifically zinc, examined the impact on nutrition outcomes of education to increase dietary diversity and improve infant feeding practices (Penny et al., 2005). The intervention group had better feeding practices, higher nutrition education levels, higher intakes of animal-source foods, and fewer children with intakes below their nutrition requirements. In addition stunting was lower and growth greater in the intervention group. Another literature review of studies of the impact of maternal education on provision of complementary foods confirmed that provision of foods and nutritional counseling resulted in increased weight and height in children (Imdad et al., 2011). Another study examined 30 peer-reviewed studies of social behavior change communication for complementary feeding practices (Lamstein et al., 2014). Almost all of the studies used interpersonal communication, while some of the studies used media, and community mobilization. Although it was not possible to measure the effect of just one intervention, the evidence suggested that multiple methods are more effective than any single method and that multiple contacts with the participants resulted in greater change. In addition, a deep understanding of the local context

58 was essential to success, which underscores the need for formative research and context assessments prior to implementation. 49 Evidence of Successful Promotion of Dietary Modification through Behavior Change Programs in Malawi In Malawi, empirical evidence suggests that the promotion of improved household processing techniques would be successful in the context of dietary preferences and women s perceptions of time. Participatory research in Southern Malawi has successfully used a community-based approach for nutritional behavior change through nutrition education and social marketing. The Tulimbe project in used an intensive community mobilization and nutrition education effort to modify food processing techniques and change consumption patterns (Gibson et al., 1998; Hotz et al., 2001; Gibson et al., 2003; Yeudall et al., 2005). The project involved an interdisciplinary team of specialists in agriculture, food and nutrition, home economics, psychology, rural extension, and community health to spread knowledge of improved food processing and feeding practices. The process focused on building relationships with community leaders, teachers, and traditional birth attendants, and spreading knowledge through the use of drama groups and plays. In one study of this project, the specific dietary interventions taught included increased production of nutrient-rich foods such as dried fish, papayas, and mangos, and soaking, germination, and fermentation to reduce the phytate content of maize and legumes. After one year, the group receiving the intervention had significantly greater knowledge of micronutrients and the food-based sources, and had greater use of fermentation of maize flour (56% vs. 1% in control group). The intervention group also had more diverse diets, lower mean phytate:zinc ratios in their diets, and a lower risk of inadequate intakes for all nutrients examined. In another study of the intervention in southern Malawi, a method of soaking pounded maize while retaining the nutritious bran was developed and taught to a group of 25 mothers (Hotz et al., 2001).

59 50 After learning about the importance of zinc nutrition, these mothers prepared and tasted the final product, and shared their perceptions of taste and cultural acceptability. A large majority of the mothers found the taste acceptable (99%), the texture acceptable (68%), and found the method culturally acceptable (96%). These findings support that when processing methods properly designed, perceptions of increased time or work pose little or no barrier to adoption. This method was specifically designed to be similar to the traditional method of maize preparation, and there were no added financial costs to the family from using this approach. Other research into consumer perception of fermented soybean in Malawi found a high degree of consumer acceptance. The study found that fermentation, including fermentation initiated through the addition of traditional thobwa beverage, greatly influenced the appearance, aromas, and flavors of soybean pastes (Tinna et al., 2013). Another study tested whether mothers in Malawi are open to feeding their children improved-formulation weaning foods based on wheat, sweet potato, and legumes (Lungu, n.d.). They found that mothers may be open to feeding the new formulations if they are deemed pleasing to the senses, underscoring the importance of addressing perceptions and food preferences. In addition to influence knowledge and perceptions, these interventions were effective in changing behaviors and practices. Another one of the Malawi studies examined a 12 month community intervention to improve education around food preparation, which including teaching soaking and fermentation techniques. They found that mothers practicing fermentation increased from 2% to 56% after the intervention, while those soaking maize flour increased from 3 to 47%, indicating a high degree of cultural acceptance (Yeudall et al., 2005). The success of these program confirms the importance of a participatory and social approach involving communication and social marketing for behavior change. Although it requires significant involvement mobilization of the local community, in the long run this approach

60 empowers communities to be self-sufficient and is more sustainable that supplementation which relies on stable infrastructure and continued financial support. 51 International Comparison of Dietary Intake and Zinc Nutrition A comparison of the dietary intake and malnutrition patterns between Malawi, Zambia, and Ghana reveals the critical role of high-phytate, maize-based diets in contributing to malnutrition in Malawi. Zambia was selected because like Malawi, maize forms a large part of the national diet and maize is not known to be processed to reduce phytate. Ghana was selected because maize also plays an important role in the diet and overall intake of animal-source food is low, however unlike Malawi and Zambia, in Ghana maize is commonly fermented to reduce phytate in such products as kenkey, banku, and gari (Table 5) resulting in higher zinc bioavailability. The same pattern is seen in Egypt where yeast-fermented (leavened) wheat bread is the staple food (Gibson and Ferguson, 1998; WHO, 2004). A study compared the zinc nutrition of rural Malawian children with Ghanaian children consuming plant-based staple food diets (Ferguson et al., 1993). The study found that while Malawian children had higher intakes of energy, protein and zinc, they also had higher phytate:zinc molar ratios. Consequently, substantially more Malawian children were severely stunted (57% vs. 28%). The study concluded that "the high intakes of phytic acid relative to zinc in Malawi suggest that these children were at greater risk for inadequate zinc nutriture than their Ghanaian counterparts" (Ferguson et al., 1993). Table 6 below displays estimates of zinc nutrition indicators for Malawi, Zambia, Ghana, and the region, based on data from FAO national food balance sheets (IZiNCG, 2004; Wuehler et al., 2005). The data are revealing. The dietary zinc and phytate-to-zinc molar ratios for Malawi and Zambia are very similar, while Zambia has a slightly higher intake of animal-source foods. Nutritional outcomes, including prevalence of zinc deficiency and stunting are also similar for the two

61 52 neighboring countries. Although Ghana has a similar daily zinc intake and only slightly higher consumption of animal products, the prevalence of stunting and the estimated proportion of the population at risk of zinc deficiency is markedly lower (48% vs. 26% and 34% vs. 21%, respectively). Part of this difference can be attributed to the lower phytate:zinc molar ratios between diets in Ghana and Malawi. Wuehler et al. (2005) determined these estimates for Sub- Saharan Africa, with and without the inclusion of West Africa. Their estimates are based on the assumption that in West Africa 100% of maize is fermented and 0% is fermented elsewhere. As expected, a much lower proportion of the population is at risk of zinc deficiency in West Africa where staples are commonly processed to reduce phytate levels than for the region as a whole. Table 6: Select indicators for zinc nutrition for Malawi, Zambia, Ghana, and regional estimates. (Source: IZiNCG, 2004; Wuehler et al., 2005). Malawi Zambia Ghana Sub-Saharan Africa, except West West Africa Only Sub-Saharan Africa, Total Africa Zinc (mg/d) ** ** 9.4 Phytate: Zinc ** ** 26.1 Molar Ratio Percent energy 2.7% 5.1% 4.3% ** ** 6.6% from Animalsource foods Prevalence of 48% 42.4% 26% ** ** - stunting Estimated % of population at risk of zinc deficiency 34% 38% 21% 35%* 18%* 28% *West Africa split from Sub-Saharan Africa on the baseline assumption that 100% of maize is fermented in West Africa, 0% in the rest of Sub-Saharan Africa. **Not provided. Conclusion Many complex factors underlie the high rates of morbidity and mortality in Malawi, which disproportionately affects women and children. Malnutrition is an underlying factor in these poor health outcomes, and zinc malnutrition in particular is involved in infection, diarrheal disease,

62 53 malaria, pneumonia, and stunting. Poor dietary quality, including low bioavailability of zinc, is due to low dietary diversity and high reliance on maize as a dietary staple. Dietary modification, through processing maize to reduce phytate using soaking, germinating, and fermenting, can increase zinc bioavailability. Behavior change communication focused on women is essential for implementing programs aimed at dietary diversification and modification to improve zinc nutrition (Brown and Hess, 2009). An intervention aimed at improving household level food processing techniques through behavior change communication and nutrition education aligns with national priorities and is contextually appropriate given the drivers of food preferences and women s decision making processes in Malawi.

63 54 3. Conceptual Framework for Research Figure 3: Conceptual Framework for Research The conceptual framework demonstrates how to the proposed research study seeks to enhance the knowledge and practice of interventions to address micronutrient malnutrition. The framework has been adapted from the UNICEF framework for the causes of malnutrition in society, which has been used for several decades of nutrition programming (UNICEF, 1990). The UNICEF framework displays three levels of the causes of malnutrition and how they ultimately lead to malnutrition outcomes. The immediate, underlying, and basic causes of malnutrition relate to the individual,

64 55 household, and community levels, respectively. The framework has been adapted here to specifically reflect zinc malnutrition, and two additional elements have been added. These are found in the boxes on the left in dotted outlines. The dotted arrows within the framework show the impact pathway from intervention to zinc nutrition outcomes. Behavior Change Impact Pathway The first of these two new elements, shown in the dotted rectangle in the upper left, demonstrates where measurement of the dietary indicator will take place in this research. The dietary indicator for this research is based on the proportion of a population with absorbed zinc intakes below the mean physiological requirement for their age and sex group. The validity of the dietary indictor has been evaluated against other methods of zinc status assessment, and has been endorsed by the WHO/UNICEF/International Atomic Energy Authority (de Benoist et al., 2007). Linkages to zinc malnutrition outcomes are shown by the dotted arrow in the upper left. The indicator does not measure functional health outcomes directly, although the linkages between absorbed zinc and functional outcomes such as stunting, pneumonia, diarrhea, and maternal health outcomes, have been previously described in the literature review. The impact of poor zinc nutrition on economic productivity and development have also been described, and the economic costs of zinc deficiency will be estimated during the analysis. The second new element, shown in the lower dotted rectangle, demonstrates where a nutrition education and behavior change intervention to improve food processing techniques would influence the causes of malnutrition. An intervention to improve food processing practices through behavior change will primarily be an education intervention. In addition to directly teaching new practices such as soaking, germinating, and fermenting, education should address cultural norms and perceptions of time, money, and effort required (Guptill et al., 1993). This intervention is expected

65 to bring about changes in food preparation practices that reduce phytate, thus improving the adequacy of dietary zinc intake as previously reviewed. 56 Whereas measurement of the dietary indicator occurs at the individual level (corresponding to the immediate causes of malnutrition) an intervention to improve maize processing will take place at the household level, corresponding to the underlying causes of malnutrition. The impact pathway through which an intervention aimed at underlying causes affects the immediate causes and ultimately the outcomes, is shown through the dotted arrows in the framework. Those boxes with thicker outlines are those elements that are directly impact along the pathway from intervention to measurement of the indicator. Health and Disease Status The disease and health status, shown in the upper-right corner of the framework under immediate causes is an important determinant of zinc malnutrition outcomes. Health status contributes directly to malnutrition outcomes, and it also influences dietary zinc intake, shown through the solid, horizontal bidirectional arrow. Malawi has an under-5 mortality rate of 71 per 1,000 live births, amounting to roughly 43,000 annual under-five deaths, 4% of which can be attributed to zinc deficiency (Black et al., 2008; UNICEF, 2014). While it has been established that zinc supplementation can reduce diarrheal episodes, there is increasing evidence that chronic intestinal inflammation, known as environmental enteropathy, can reduce the ability of the gut to absorb nutrients. Environmental enteropathy exacerbates zinc deficiency through reduced absorptive capacity of the intestine, while zinc deficiency can worsen enteropathy through several pathways including systemic inflammation, thus underscoring this bidirectional pathway (Lindenmayer et al., 2014). Therefore, it must be emphasized that an intervention to increase zinc absorption does not exist in a vacuum, but rather should be part of a

66 wider approach in improve the health and disease status of children, pregnant and lactating women, and other vulnerable groups through health systems and food systems approaches. 57 Institutional Environment for Health and Education The behavior change intervention to increase zinc bioavailability takes place in the broader context of the institutional environment in Malawi. This is shown in the framework below Information and Education as on the level of Basic Causes. The institutional context, which has been reviewed previously, includes national bodies such as the Department of Nutrition, HIV, and AIDS, international initiatives such as the Scaling up Nutrition movement, donors such as USAID, NGOS such as Concern Worldwide, and national policy guiding frameworks. The National Nutrition Policy and Strategic Plan prioritizes maternal nutrition and care, improved young child feeding practices, and improved intake of micronutrients. In addition, the National Nutrition Guidelines promote processing methods such as germination and fermentation to improve the nutrition of foods and prioritize behavior change and education as impact pathways to reduce stunting. The institutional context is important for evaluating the potential of an intervention to improve zinc deficiency, and it is also important for comparing feasibility and cost-effectiveness between intervention options and ensuring that those options align with national priorities. Assumptions and Limitations of the Framework Several simplifying assumptions must be made in this framework. An assumption will be made that other elements of the framework, including household access to food, the political and economic environment, and the access to health services, do not change as a result of the intervention. The wider context, which has been described in the background section of this paper, is assumed not to change for the purposes of analysis. There may be benefits from the promotion of household processing methods besides increased dietary zinc that are not captured in this framework. These may include women s empowerment through increased education, promotion of

67 58 local knowledge, and improved gut health as fermentation may reduce the incidence of diarrhea through the prevention of contamination. These additional benefits have been reviewed in the previous literature review section.

68 59 4. Research Questions Research Question 1a: What is the current estimated prevalence of zinc deficiency in Malawi for population subgroups of interest? Research Question 1b: How do the estimated prevalence levels compare across regions and between urban and rural residence? Research Question 2a: What is the expected reduction in the prevalence of zinc deficiency resulting from the adoption of improved household maize processing techniques? Research Question 2b: How do these changes vary across regions and between urban and rural residence? Research Question 3a: What is the expected reduction in the prevalence of zinc deficiency from an alternative intervention based on biofortification? Research Question 3b: How does an intervention to promote improved food processing compare to biofortification at the national level at various levels of coverage? Research Question 3c: How does an intervention to promote improved food processing compare to biofortification for each geographical region?

69 60 5. Methodology 5.1 Assessment of Prevalence of Zinc Malnutrition in Malawi and Simulation of the Impact of Food Processing and Biofortification This study answers the above research questions using a quantitative approach to assess the population prevalence of zinc deficiency risk in Malawi, and to estimate the change in prevalence through the introduction of processed (soaked, germinated, and fermented) and biofortified maize. This study used household level food consumption data from the 2011 Malawi Third Integrated Household Survey. Intra-household food allocation was assumed to be proportional to the energy needs of individuals according to age and sex, and the concept of adult equivalent units was used to derive a household size based on these energy needs (Weisell and Dop, 2012). Using published food composition data, the total apparent phytate and zinc daily intakes for each individual in the household were calculated. Next, using the mathematical model for zinc absorption as a function of total zinc and phytate published by Miller et al., (2007), the total apparent absorbed zinc was calculated for each individual in the sample for those age groups for whom the equations are considered valid. The final step in the initial assessment involved determining the population risk of zinc deficiency using the Cut-Point method (Institute of Medicine, 2000). The assessment was performed at the national level for each physiological group and was then disaggregated based geographical region and urban or rural status. After this baseline calculation, a simulation of the proposed intervention was performed to reflect a 50% reduction in phytate from household processing in maize-based foods (Hotz, Gibson, and

70 61 Temple, 2001). This simulation reveals the change in population prevalence of the risk of zinc deficiency if all households were to adopt the new technology; it was then repeated with a random selection of 40%, 20%, and 10% of households. Next, the simulation was repeated for biofortification, increasing the total zinc levels to those expected to result from maize biofortification, and testing that intervention at varying levels of coverage. Data cleaning and handling, variable creation, and statistical tests were completed using STATA 12.0 software. All means and statistical tests were computed using the surveys weights, and the SVY module of STATA was used for to account for the probabilistic nature of the survey design. The mean amounts of absorbable zinc for each physiological group were compared between urban and rural areas using Student s t-test, and between the three geographical regions using analysis of variance (ANOVA) followed by Tukey post-test. Significance was tested at the p<0.001 level for all comparisons. The prevalence rates of inadequate zinc intake were determined using the cumulative distribution function (CDF.NORMAL) in IBM SPSS Statistics Data Sources, Cleaning, and Preparation The household food consumption data for this study was gathered through the Republic of Malawi s Third Integrated Household Survey , released in the spring of 2012 (World Bank, n.d.). The Survey is conducted by the National Statistics Office every five years, with the present data collected during a period from The program was supported by the World Bank Living Standards Measurement Study Integrated Surveys on Agriculture (LSMS-ISA) Initiative. The total survey sample includes 12,271 households across the three regions in Malawi: North, Central, and South, and used a stratified, two-stage cluster sampling design. The primary sampling units for the first stage of the selection process were the 768 Enumeration Areas previously defined from the 2008 Population and Housing Census. Enumeration Areas were systematically selected

71 62 with probability proportional to size within district, based on the number of households in each area. In the second stage of selection, households and replacement households were selected randomly to ensure adequate sample size. The sample of 12,271 households is representative at the district level. A household s status as urban or rural was defined in the survey and a variable was included in the data set to mark residence. The urban regions include the four major urban areas (Lilongwe City, Blantyre City, Mzuzu City, and Zomba), while other areas are considered rural. Geographic residence (north, central, or south) was based on district of residence, which was classified into one of three official regions in the Basic Information Document of the Malawi Third Integrated Household Survey. Household Roster The table below displays the number of household members belonging to each group according to their age and sex group based on zinc mean physiological requirement (MPR), by region and urban status, in the Malawi Third Integrated Household Survey. These age and sex groups represent the groups used for the analysis of the prevalence of zinc deficiency in the study. Table 7: Number of Household Members Belonging to Each Group, by Region and Urban Status Group based on Zinc Pop North Central South Urban Rural Mean Physiological Requirement Age 6-11 months Age 1-3 years Age 4-8 years Age 9-13 years Age years (males) Age years (females) Age >18 years (males) Age >18 years (females) Women of childbearing age (14-49 yrs)

72 63 The survey accounts for people that are not listed as family members but who did eat meals at the household. These guests were appended into the data set, and a portion of the weekly food was assigned to them based on how many meals they ate in the house. However, because their exact age and sex group was not recorded, they were not included in the MPR groups for determining zinc deficiency prevalence. Ultimately, they represented a very small portion of the total (<0.4%). The survey accounts for household members that may be absent (from migration or otherwise), as it records how many days each member ate any meals in the house within the past 7 days. The vast number of household members included in the survey, 53,624 or 95%, ate in the household all seven days. Only 1,097 people (1.94% of total) ate zero meals in the previous seven days and were not included in the analysis. Respondents eating only some of the days represented a small fraction of the total; those consuming two days or less were dropped form the analysis while those consuming at least 3 days were included. The MPR group for women of child-bearing age was defined as women aged (Engle-Stone et al., 2014). Status as pregnancy or lactating was not recorded in the survey, so it was not possible to establish the proportion of women in each physiological state meeting their zinc requirements. However it is possible to determine the proportion of total women of childbearing age who meet the requirements of pregnancy and lactation, and to interpret that figure appropriately. The Estimated Average Requirements for children under 2 are based on assumptions that children receive a standard amount of breastmilk, and they therefore represent only dietary requirements. Infants under six months are not included in the assessment of zinc deficiency based on dietary intake because the International Zinc Consultative Group has determined that breastmilk is a sufficient source of zinc for exclusively breastfed, normal-birthweight, term infants until about 6 months of age (IZiNCG, 2004).

73 64 Preparation of the food consumption data from survey The food consumption data from the present study comes from the food consumption module, which asks household respondents about food groups and amounts consumed over the past one week. This dataset includes 124 food items across 11 categories, a sample of which is shown in the Table below. Table 8: Examples of Food Categories and Foods Survey in the Malawi Third Integrated Household Survey Food Category Examples of Foods Surveyed Cereals maize flour (normal, refined, bran); green maize; rice; finger millet; sorghum; pearl millet; wheat flour; bread; buns; biscuits Tubers Cassava; cassava flour; sweet potato (white, orange); Irish potato; plantain Nuts and pulses Vegetables Animal products Fruits Cooked foods from vendors Others beans (white and brown); pigeon pea; groundnut; soyabeans onion; cabbage; wild greens; cucumber; pumpkin; okra; mushroom eggs; fish (dried, fresh); beef; goat; port; chicken; milk; butter; cheese mango; banana; citrus; pineapple; guava; apple; wild fruit maize; chips; cassava; eggs; chicken; fish; samosa sugars; oil; fruit juices; traditional beer and drinks; spices; jams; sweets The food consumption module of the Malawi Third IHS measures total consumption, which includes purchases, own-production, and gifts. The enumerator manual specificly instructs enumerators to record only the quantity of food consumed, not expenditures. The consumption module includes food received as gifts, cooked food from vendors, as well as other food consumed outside the household by any household members. Any other food that was purchased but not consumed, for example saved to the pantry, fed to animals, or lost to spoilage are not recorded in the food consumption survey. The assumption is therefore that the recorded quantity is only the quantity consumed, and does not include food waste nor non-edible portions that were discarded. The survey measures food consumption using 23 different units, ranging from standardized measures such as kilograms to non-standardized measures such as heaps, pails, and basins. A photo aid was provided to the enumerators to assist the respondent in identifying the amounts of food

74 65 consumed, and a supplemental market survey was conducted to identify standardized conversion factors to convert these quantities into kilograms. These conversion factors were included with the original data set. A number of measures were taken to clean the data including identifying and dropping uninterpretable coding errors and converting those units that were specified incorrectly. For some unit-food combinations, conversion factors were not provided; these units were therefore changed to their closest approximations to estimate conversion factors or were dropped if necessary. Ultimately, these were a small fraction of total food intake responses (0.11%). The conversion factors were then merged into the dataset to allow conversion of all quantities to kilograms. Estimating Intra-Household Food Distribution Using Adult Equivalence Units The Malawi Third Integrated Household Survey uses the household rather than the individual as the sampling unit. Therefore, some assumptions must be made to estimate the intra-household allocation of food in order to estimate individual intakes (Deaton, 1997). The adult equivalence unit (AEU) concept can be used to specify intra-household food distribution. It is based on the assumption that each individual s share of household food is determined by their relative energy need, according to their age and sex (Fiedler et al., 2012; Weisell et al., 2012). This approach assigns an arbitrary reference group (in this case a year-old male) a value of 1 AEU Unit, and assigns all other household members a value equal to the ratio of their energy needs to those of the adult male reference group. These values are based on the Food and Agriculture Organization (FAO) age- and sex-specific estimates of individual energy requirements. Individual AEUs are summed to reach the total household AEUs. The amount of each food item consumed by the household is then first divided by the total household AEUs, then multiplied by each member s fraction of an AEU unit (Murphy et al., 2012). Using this method of food allocation, the nutrient consumption of each individual in the household can be estimated based on apparent intake.

75 66 The assumption that food is distributed proportionally to energy needs is reasonable for planning and evaluating between interventions, as data suggests that on average, groups receive dietary energy within 20% of estimates based on their needs (Berti, 2012). However violations of these assumptions inevitably effect the predicted nutrient intake, and so the effect of changing these assumptions are discussed during the analysis. Identification of Outliers The data on household-level food consumption of each food group was analyzed for outliers, and outliers for households that reported eating unrealistic amounts were cleaned from the data set. The total value of AEU for each household was calculated from the household roster as noted above. The total weight of each food was then divided by AEU, and this value (kg/aeu) was examined. Outliers were defined here as those observations exceeding the 75th percentile by more than 3 times the inter-quartile range, and represented approximately 3% of observations (Dary & Imhoff- Kunsch, 2012). The values of outliers were replaced by the mean value for that food item. 5.3 Preparation of Malawi Food Composition Table A food composition table was compiled containing the phytate and zinc levels for all of the foods in the Malawi Third Integrated Household Survey data set. The sources of food composition data are listed below according to the order in which they were consulted. Despite the increasing availability of food composition data and improved methods for measuring nutrient levels, food composition data represents an estimate which varies based on local conditions, storage, and preparation practices. Every effort was made to use data that was appropriate for Malawi when available or that has been used in other research. The levels of zinc and phytate were defined in milligrams of the nutrient per 100 grams of food. Nutrient retention during cooking as well as the density of liquids when converting from volume to weight were accounted for during the data analysis. Neither zinc nor phytate levels are expected

76 67 to be greatly affected by heat during cooking. According to the USDA Table of Nutrient Retention Factors (2007), the retention factor for zinc is nearly 100% for nearly all foods that are not cooked in water. For most vegetables, legumes, and meats cooked in water the retention factor is 95%. Additionally, whether or not cooking water is retained and consumed was not specified in the Malawi IHS3. For these reasons no adjustment for zinc or phytate loss during cooking was made in the data. Thirty-five foods with no zinc and phytate were dropped from the data set. The following sources were used for food composition data: Gibson, R. S., & Ferguson, E. L. (2008). An interactive 24-hour recall for assessing the adequacy of iron and zinc intakes in developing countries. Washington, DC: HarvestPlus. Tanzania Food Composition Tables. (2008). Harvard School of Public Health, Boston. US Department of Agriculture (USDA) Nutrient Database for Standard Reference, Release 28. Washington, DC. Loveness K. Nyanga, Tendekayi H. Gadaga, Martinus J.R. Nout, Eddy J. Smid, Teun Boekhout, Marcel H. Zwietering, Nutritive value of masau (Ziziphus mauritiana) fruits from Zambezi Valley in Zimbabwe, Food Chemistry, Volume 138, Issue 1, 1 May 2013, Pages International Network of Food Data Systems (INFOODS) Database, Food and Agriculture Organization. World Food Programme Food Composition List. Food Aid Information System Calculation of Zinc Nutrition Indicators and Total Absorbed Zinc Calculating Individual Estimated Absorbed Zinc Using the Miller Equations The food composition data was merged into the data set, and total dietary phytate and zinc were summed across food items at the household level for the seven-day period. The quantities were then divided according to estimates of intra-household distribution described previously, and divided by seven to yield estimated individual zinc and phytate intake per day. Dietary indicators

77 of zinc nutrition, including mean dietary zinc, mean dietary phytate, and mean phytate:zinc molar ratios were calculated for each group. 68 The total absorbable zinc can be estimated per individual using bioavailability algorithms provided by Miller et al. (2007), and updated by Hambirdge, et al. (2010), as presented in the literature review. As previously described, these algorithms provide an improvement over the use of blanket absorption rates for entire age and sex groups, and this is the approach recommended by HarvestPlus in their calculations of target levels for biofortification (Fairweather-Tait et al., 2012). As described in the literature review, there is insufficient evidence to apply the Miller model of zinc absorption to very young children, and consistent with other studies, this was not done in the present analysis (Arsenault, 2010). Therefore only total dietary zinc, rather than absorbed zinc, was determined for these groups. Statistical Tests After absorbed zinc was calculated at the individual level, the weighted group means and 95% confidence intervals were calculated for all physiological groups at the nation level and for geographic regions and urban residences. The distributions of absorbed zinc were tested for normality visually and using statistical normality tests. Although they passed visual inspection, several distributions did not pass statistical tests, most likely because these tests are overly sensitive at the very large sample sizes encountered in this study (Gibson and Ferguson, 2008). Moreover the statistical tests used to compare means (Student s t-test and ANOVA) are robust to modest violations of the normality assumptions at large sample sizes, and thus were considered valid for the present study. A Student s two sample t-test was performed for urban and rural physiological groups to test significance of difference of the means. For the three regions, means were compared using analysis of variance (ANOVA) followed by a Tukey test between each pair. Significance was tested at the

78 p<0.001 level for all comparisons. These results of significance tests are indicated in the tables in the statistical appendix for each model Calculation of the Prevalence of Inadequate Zinc Intakes Method Overview An estimation of the prevalence of inadequate zinc intakes was made using the cut-point method for population nutritional assessment described by the Institute of Medicine (2000). This method uses several statistical techniques to estimate the population prevalence of the risk of inadequate intakes based on the distribution of individual intakes. The cut-point approach estimates the proportion of the population with intakes below a threshold, such as the Estimated Average Requirement or Mean Physiological Requirement. That proportion represents the prevalence of risk of inadequate intakes for that population. Although the EAR is commonly used, the mean physiological requirement (MPR) was used in this study. It has been successfully used in several studies and has certain advantages (Wuehler et al, 2005; Denova-Gutiérrez, et al., 2008; Bermudez et al., 2012). While the EAR assumes an average bioavailability of zinc for entire population groups, using the mean physiological requirement allows estimating absorbed zinc for each individual, based on both zinc and phytate intakes (Miller et al., 2007; Hambidge et al., 2010). In addition to being a more precise estimation of zinc bioavailability, this approach permits new estimations of zinc absorption resulting from the reduction of phytate through processing, because new absorption rates will result from phytate reduction even without increases in dietary zinc. The Cut-Point method is valid when several conditions are satisfied. It is most accurate when the population prevalence approaches 50%. The intake of the nutrient and the requirement must be independent; that is, the intake must not be correlated with the requirement, as it is for energy.

79 70 Additionally, the method assumes that the distribution of nutrient requirements is symmetrical and that the variance of intakes is greater than the variance of requirements (Institute of Medicine, 2000). These assumptions are valid for zinc assessment (IZiNCG, 2004). Based on the above methodology, a baseline estimate of the prevalence of inadequate zinc intakes was determined for each of the MPR groups described above. The prevalences of inadequate zinc intake were determined using the cumulative distribution function (CDF.NORMAL) in IBM SPSS Statistics 22. The mean physiological zinc requirement was applied to each age and sex group as the cut-point. The area under the curve and below the cut-point was reported as the proportion of the population with intakes below their requirement. Estimates of prevalence of zinc inadequacy for physiological groups were then disaggregated by urban/rural status and geographical region of residence in (Northern, Central, or Southern regions). All results are presented in the tables in the appendix. Estimating the Width of the Distributions of Absorbed Zinc (Coefficient of Variation) During the period covered in the Malawi Third Integrated Household survey, some households will have consumed more and other less than usual. Although the mean is considered a valid estimate of the population mean, the width of the intake distribution will be wider than the width of distribution of usual intakes (Murphy, et al., 2012). The observed distribution of intakes must therefore be adjusted to remove day-to-day intra-individual variation and preserve only interindividual variation in zinc intake. The adjustment process yields a distribution with the same shape as the original, but with reduced variability (IZiNCG, 2004). In studies where multiple data points are collected for each respondent, several statistical approaches can be used for adjusting the nutrient intake distributions. However as only one data point is available in the current study, an estimate of the coefficient of variation must be derived from the information on the width of adjusted intake distributions for other populations. Using

80 unadjusted intakes severely biases results, and research shows that applying external variance estimates greatly improves the accuracy when compared to no adjustment (Jahns, 2005). 71 The International Zinc Consultative Group recommends apply a coefficient of variation of 25% for dietary zinc intake in studies containing only one point of recall data, and this has been used in numerous nutritional assessments (Wuehler et al., 2005; Hotz, 2007; Denova-Gutiérez et al., 2008). However this figure is for zinc intake rather than absorbed zinc. The distribution of absorbed zinc in the population is likely to be narrower than the distribution of zinc intakes, because large changes in zinc intake result in smaller changes to absorbed zinc due to the saturation-response model described by Miller et al. (2007). Estimates of the coefficient of variation of absorbed zinc in a population were not available, although it would follow that a figure lower than 25% should be used. The 25% coefficient of variation does appear to have been applied to absorbed zinc in Wessells et al., (2012), although there is reason to believe its use will lead to biased results. Using an adjusted coefficient of variation figure of 25% is not considered to be valid when the observed coefficient of variation is already less than 25%. This was the case with most of the distributions in the current research (IZiNCG, 2004). Therefore this study also examined the impact of using multiple assumptions of the coefficient of variation to predict the prevalence of zinc deficiency. Estimates were made using the figures of 25%, 20%, 15%, and 10% for the coefficient of variation. For reporting the results, a coefficient of variation of 20% is assumed unless otherwise specified, and all results are presented in the tables in the appendix. The effects of changing the coefficient of variation on the predicted population prevalence are discussed in the sensitivity analysis section in the conclusion.

81 72 Model Uncertainty and Sensitivity Analysis As with all theoretical models, there is uncertainty arising from the specification of the model and from choice of parameters used as inputs. Uncertainty in this analysis was addressed through the procedures for sensitivity analysis described by Walker and Fox-Rushby (2001). The main uncertainty in this model arose from estimates of the width of the distribution of zinc intakes and from the level of coverage of the different intervention options (described below). The range of values for the coefficient of variation was determined based on the estimates from the literature as described. The selection of values to test for the coverage parameter was made based on a maxmin analysis combined with an optimistic-pessimistic scenario (Walker & Fox-Rushby, 2001). The values selected are described in the simulation methodology below. 5.6 Simulation of Household Food Processing Estimating the change in prevalence of zinc deficiency from improved household maize processing methods The household processing methods of soaking, germinating, and fermenting increase the bioavailability of zinc in the diet through the reduction of the inhibitor phytate. Phytate is the most important inhibitor and one of the principal determinants of zinc absorption in the diet (Miller et al., 2007). Reduction of phytate through household processing occurs through both the passive diffusion of phytate into the soaking water, or through enzymatic hydrolysis of phytate during germination and fermentation. This simulation estimated the change in population zinc deficiency prevalence through the reduction in phytate in maize processed at the household level. The amount of phytate reduced through these processing methods can vary depending on many conditions including time, temperature, and the substrate. For this study, a 50% reduction in the phytate content of Malawian maize consumed at the individual level was assumed. This figure is consistent with the

82 73 experimental data in the literature and has been used as a baseline assumption in other studies (Gibson et al., 1998). Because maize is the principal staple in Malawi and because phytate is largely responsible for the low zinc bioavailability in diets, it was hypothesized that the change would have a substantial impact on population zinc risk prevalence. To run the simulation, the levels of phytate in maize products in the food composition database were reduced by 50%. These foods included Maize ufa mgaiwa (normal flour), Maize ufa refined (fine flour), Maize ufa madeya (bran flour), Maize grain (not as ufa), and Green maize. Using the zinc bioavailability algorithms proposed by Miller et al. (2007), new estimates of individual absorbed zinc were estimated. Weighted means of absorbed zinc were computed for adult age-sex groups, and prevalence rates were calculated under various assumptions of the coefficient of variation. Prevalence rates were also calculated based on geographic region and urban residence. Because the process occurs at the household level and does not target specific sub-groups, all individuals were assumed to consume the improved maize product at the same rate as current maize consumption. The initial simulation was performed at a hypothetical 100% coverage to establish a maximum upper bound. In the subsequent simulations, 40%, 20%, and 10% of households were randomly selected to simulate partial coverage of the intervention. These coverage values were based on the estimated adoption rates of improved maize in Africa (Hotz, 2009; Meenakshi et al., 2010). Forty percent and 20% were considered optimistic and pessimistic coverage scenarios, respectively, while 10% was tested as a minimum lower bound. The International Zinc Nutrition Consultative Group suggests that the risk of zinc deficiency is a public health concern when population prevalence exceeds 25%. Prevalence rates were evaluated against this benchmark in the results section below.

83 Simulation of Biofortification Estimating the change in prevalence of zinc deficiency from increased zinc levels through biofortification of maize Biofortification can increase the nutrient levels in staple food crops through both conventional and transgenic breeding methods (Saltzman et al., 2012). Increased zinc levels have been achieved in crops such as pearl millet, beans, rice, and wheat, which have been successfully disseminated in Africa, while maize varieties have been successfully fortified with Provitamin A Carotenoids and used in Zambia, Nigeria, Brazil, China, and India (Saltzman et al., 2012). An experimental high kernel-zinc maize variety, being developed by the International Maize and Wheat Improvement Center, has zinc levels of 3.4 mg/100 grams (Chomba et al., 2015). A study comparing this variety to a traditional variety in Zambia found that absorbed zinc was significantly greater among a randomly-selected group of children consuming the biofortified variety over the traditional variety (Chomba et al., 2015). Biofortification strategies are not expected to reduce phytate levels, as low phytate maize varieties were shown to have reduced crop yields and are therefore unlikely to be adopted (Bregitzer et al., 2006). This simulation determined the change in the prevalence of zinc deficiency for each physiological group from the adoption of this new, high-zinc maize variety in Malawi. To perform the simulation, the original food composition table was adjusted to reflect the new maize zinc levels (3.4mg zinc/100 grams). As in the simulation of household processing, the initial simulation was performed at a hypothetical 100% coverage to establish a maximum upper bound. Coverage of 10% was used a minimum lower bound, and 40%, 20% coverage was simulated under an optimistic-pessimistic scenario for adoption of biofortified maize in Africa (Hotz, 2009). The new prevalence rates were computed for each physiological group at the national and subnational levels, for each assumption of the coefficient of variation. The analysis of the impact of

84 75 biofortification included children because higher absorbed zinc would result from higher dietary zinc in the model. The resulting prevalence rates were compared against both the 25% threshold for public health concern, as well as the rates of equivalent coverage of the household processing techniques. 5.8 Validity and Limitations of the Methodology Validity of the Dietary Indicator for Zinc Assessment Dietary intake is one of several measures for assessing zinc status at the population level, along with blood serum zinc concentration and stunting prevalence (Gibson et al., 2008). The dietary indicator has several limitations, including its reliance on assumptions about intra-household food allocation and nutrient composition. Its validity is also dependent on the accuracy of calculated rates of nutrient absorption and nutrient requirements, which may require future revision. There is recent evidence that the dietary indicator may overstate zinc intake, and thus under-estimate deficiency, in children. Nevertheless, the validity of the dietary indictor of estimates of population prevalence from intake data has been evaluated against other methods of zinc status assessment, and was endorsed by the WHO, UNICEF, and the International Atomic Energy Authority, concluding that reported prevalence of low serum zinc concentration and the estimated prevalence of inadequate zinc intakes predict similar levels of risk of zinc deficiency (Hotz, 2007). The dietary indicator is considered valid for planning, assessing populations and subgroups, and evaluating the potential changes in zinc deficiency prevalence after the introduction of fortified foods (Denova-Gutiérrez et al., 2008; Harvey et al., 2010; Arsenault et al., 2010). Limitations of Using Household Consumption and Expenditure Surveys There are also limitations to using household consumption and expenditure surveys (HCES), such as the Malawi Third Integrated Household Survey, for nutrition analysis. Unlike 24-hour food recall

85 76 surveys or weighted food records, which are typically considered the gold standard for food consumption data, HCES typically measure only apparent food consumption. However, one of the strengths of the Malawi Third Integrated Household Survey is that it does in fact record only food consumed by household members, including food consumed outside of the house, wild foods, food consumed from existing inventory, or received as gifts. The survey excludes food that was purchased but went to waste, was stored, or was fed to animals. Misspecification of the units, as well as non-standardized units such as bunches and heaps introduce potential error, and assumptions had to be made when preparing the data. However the methodology used for data preparation does not result in any bias that would systematically overor underestimate food intake. Nevertheless, household consumption and expenditure surveys hold a number of advantages, as they are widely available and less costly than 24-hour survey data. The cost of 24-recalls can be up to 75 times higher than the cost of analyzing existing HCES data (Fiedler et al., 2013). Thus in most cases, the trade-off is not between the two types of surveys, but rather between having the less precise HCES data or having no nationally representative data at all. The fact that they are nationally representative is a major strength of HCES, and allows analysis of different population strata which traditional dietary surveys cannot because of limitations on sample size. Not all programmatic decisions require a so-called gold standard, but rather the assessment should be selected based on the degree of confidence needed in the result. Bearing this in mind, HCES offer the greatest suitability at the lowest cost for estimating the risk of inadequate intakes and estimating the coverage and impact of interventions, and to compare between options (Coates et al., 2012). Consumption and expenditure surveys may in fact be more accurate at the population level because they include data for the entire household not just women and children (Jariseta et al., 2012).

86 77 Additionally, 24-hour recalls often introduce recall bias up to 20% of intake (Murphy et al., 2012). Studies comparing the two methods have found high level of consistency between 24 hour recalls and HCES for the most commonly consumed items, and no statistically significant difference in estimated zinc intakes (Fiedler et al., 2012; Jariseta et al., 2012).

87 78 6. Primary Research Findings: Simulation Results 6.1 Research Question 1 Research Question 1a: What is the current estimated prevalence of zinc deficiency in Malawi for population subgroups of interest? Research Finding 1a: The national prevalence of zinc deficiency is very high among most physiological groups, including infants and women of childbearing age. The estimates of adult women at risk range from 23-34%, while less than 4% of women of child bearing age meet the zinc requirements for breastfeeding. Over 83% of children under one year are at risk of inadequate zinc intakes. Children aged 1-13 years had a lower-than-expected risk prevalence, which may be due to the method used for assessment. Over 70% of men had absorbed zinc levels below their mean requirements. Women Table 9: National Indicators of Zinc Nutrition for Females Group 6: Group 8: Females Age Females Age > 18 Group 9: Females of Child-bearing Age (Pregnancy MPR) Group 9: Females of Child-bearing Age (Lactation MPR) Mean Physiological Requirement, (mg/day) Mean Absorbed Zinc (95% confidence 2.16 ( ) 2.19 ( ) 2.20 ( ) 2.20 ( ) interval) (mg/day) Population Below MPR (20% CV) 34.03% 22.58% 86.49% 96.29%

88 79 The nutritional analysis revealed that a large portion of Malawian women from all age groups are at risk of inadequate zinc intake. Table 9 displays the national estimates of the prevalence of inadequate intakes for women aged 14-18, those over 18, and those of childbearing age. At the population level, females aged had a mean absorbed zinc level of 2.16 mg/ day. Applying the cut-point of the mean physiological requirement of 1.98 mg/day at an assumed 20% coefficient of variation, 34.03% of young women were at risk of inadequate intakes. At other assumptions of the coefficient of variation, this figure ranged from 20.52% to 37.1%. Tables containing estimates resulting from other coefficients of variation are provided in the appendix. For women over age 18, the mean absorbed zinc was 2.19 mg/day, compared to a mean physiological requirement of 1.86 mg/day. At a 20% coefficient of variation, 22.58% of women over 18 were at risk of deficiency. This figure ranged from 6.61% to 27.35% under different assumptions. The data set does not indicate whether women were pregnant or lactating. However the number of all women of childbearing age who meet the zinc physiological requirements for pregnancy or lactation can still be observed. Depending on the assumptions for the coefficient of variation, between 86 and 98% of Malawian women of childbearing age failed to meet the mean zinc requirements for pregnancy, while 96 to 99% failed to meet the requirements of lactation. Although only a portion of these women are actually pregnant or lactation, these figures suggest nevertheless that few of those women meet their requirements. Men Table 10: National Indicators of Zinc Nutrition for Males Group 5: Males Age Group 7: Males Age > 18 Mean Physiological Requirement (mg/day) Mean Absorbed Zinc (95% confidence interval) (mg/day) 2.26 ( ) 2.32 ( ) Population Below MPR (20% CV) 71.57% 78.44%

89 80 The zinc nutritional analysis for adolescent and adult men agrees with the baseline findings for women that zinc deficiency rates are problematically high in Malawi. For males aged 14-18, the mean absorbed zinc of 2.26mg/day falls below the mean physiological requirement for that age group of 2.52mg/day. The proportion of males age with at risk of inadequate absorbed zinc ranged from %, and was 71.57% at an assumed 20% coefficient of variation. For Malawian men over age 18, despite their higher estimated absorbed zinc levels of 2.32 mg/day, the majority still fell below the MPR of 2.69 mg/day. Depending on the assumptions, the proportion at risk of inadequate intakes ranged from % nationally, and was 78.44% at the 20% CV. Because the mean intake for men falls below the MPR unlike for women, lower estimates of the CV increase the proportion at risk. A comparison of these estimates are provided in the appendix and the effect of changing the CV is explored in the section on sensitivity analysis. This baseline nutritional analysis indicates extremely high rates for the prevalence of inadequate absorbed zinc among the general population of Malawian men. Children Table 11: National Indicators of Zinc Nutrition for Children Children Age 6-11 months Children Age 1-3 years Children Age 4-8 years Estimated Average Mean Physiological Requirement Mean dietary zinc (95% confidence interval) (mg/day) Population Below EAR (25% CV) 3.23 ( ) 4.88 ( ) 6.52 ( ) 82.97% 0.91% 6.09% Requirement Mean Absorbed Zinc (95% confidence interval) (mg/day) Children age 9-13 years ( ) Population Below MPR (20% CV) 9.38% Eighty-three percent of children between 6 and 12 months of age were found to be at risk of inadequate dietary zinc intakes, based on total dietary intake, the Estimated Average Requirement, and a coefficient of variation for dietary intake of 25%. Estimates using other CVs were not performed because 25% is the recommended figure to use for dietary zinc, as opposed to absorbed

90 81 zinc, which cannot be predicted using the Miller equations for children (IZiNCG, 2004). For older physiological groups of children, the resulting proportion at risk was substantially lower than expected. For children 1-3 years of age, only 0.91% were at risk of inadequate intake according to the baseline dietary analysis, and the corresponding rates were 6.09% for children 4-8 years, and 9.38% for children 9-13 years. These prevalence rates for children over one year-old were lower than expected based on a 47% national stunting rate and the high corresponding deficiency rates for adults. It is generally accepted that the prevalence of micronutrient deficiency for children is higher than for adults because of the greater nutrient density requirements of children (Wessells, 2012). The possible reasons for these lower-than-expected prevalence rates and implications for the research are discussed in later sections. Research Question 1b: How do the estimated prevalence levels compare across regions and between urban and rural residence? Research Finding 1b: The South had the highest baseline prevalence of inadequate zinc intake, followed by the North and Central regions. Although dietary maize intake was slightly higher in the Central region, the South had the highest phytate to zinc molar ratio of the diet and the highest levels of stunting. The prevalence of deficiency and stunting rates were higher, and zinc absorption lower, in rural areas than in urban areas. Dietary Maize, Zinc, and Phytate Intake Table 12 below shows several dietary indicators of zinc nutrition at the national and sub-national levels. The average daily maize consumption per energy requirement was highest in the Central region, followed closely by the South, while the North was 25% lower. Rural areas consumed 20%

91 82 more maize than urban areas, and all differences in means were statistically significant. The pattern of dietary zinc intake did not follow the pattern of maize consumption. Zinc intake was higher in urban areas compared to rural, and was highest in the South, followed by Central, then the North. Higher dietary zinc intake may be due to either increased cereal staples or animal products. However the pattern of total dietary phytate is indicative of greater reliance on cereal staples. Unlike zinc, the pattern of phytate intake nearly followed the pattern of maize consumption: phytate intake was lower in urban areas than in rural, and was 40% lower in the North than in the South. Table 12: Indicators of Zinc Nutrition, Regional and Urban Disaggregation Population North Central South Urban Rural Mean maize intake a 0.61 b 0.58 c * per AEU (kg) (per day) Mean dietary zinc a 9.11 b c * intake (mg) Mean dietary a b c * phytate intake (mg) Mean Phytate: Zinc a b c * Molar Ratio Mean Absorbed a 2.30 b 2.19 c * Zinc (mg) (age >14) Mean Percentage of 23.97% 29.34% a 25.07% b 21.99% c 24.37% 23.89% dietary Zinc Absorbed (age >14) Under 5 Stunting Prevalence 47% 45% 47% 48% 41% 48% a,b,c Indicates difference in means between regions is statistically significant at the p<0.001 level. *Indicates difference in means between rural and urban is statistically significant at the p<0.001 level. Zinc Bioavailability The mean phytate to zinc molar ratio was highest in the South, followed by Central, and lowest in the North, and comparisons between all regional means were statistically significant. Rural areas had statistically significantly higher average molar ratios than urban areas. These findings suggest a lower zinc bioavailability in the South and in rural areas. There was a small, but statistically significant difference in mean absorbed zinc, predicted by the Miller equations, between the Central

92 83 and the North, while the average absorbed zinc in the South was substantially lower. As expected, average absorbed zinc was higher, by 9%, in urban than rural areas. As a percentage of dietary zinc consumed, absorbed zinc was over 7% higher in the North than in the South, as expected from the lower molar ratio in the North. Under 5 Stunting The pattern of under-5 stunting prevalence follows the pattern of zinc bioavailability predicted by both the molar ratio and the Miller equations. Like the molar ratio, stunting prevalence was highest in the South, followed by the Central, then the North, and was higher in rural than urban areas. Table 13: Regional Comparisons of Zinc Deficiency Prevalence North Central South Urban Rural Children 6-11 mo % 86.08% 76.46% 86.26% 82.39% Children % 1.00% 0.73% 0.88% 0.91% Children % 7.07% 4.62% 5.41% 6.21% Children % % % 5.60% 10.20% Women % % % 23.84% 36.41% Men % % % 59.08% 73.76% Women > % 19.79% 26.07% 14.24% 24.60% Men > % 74.37% 83.15% 65.11% 81.13% Women of CBA*, pregnancy 85.32% 83.47% 89.56% 74.80% 88.57% Women of CBA*, lactation 95.80% 94.96% 97.49% 90.37% 97.12% + Difference in mean absorbed zinc between North and Central for these groups was not statistically significant. *CBA: Child-bearing Age. The table above displays the predicted baseline prevalence of zinc deficiency for all physiological groups across geographical and urban regions, at a 20% coefficient of variation. Several important patterns emerge from analysis. For all adult groups to which the Miller equation was applied and the MPR used as the cut-point, the South had the highest proportion of the population at risk of zinc deficiency. Estimated zinc deficiency rates in the North and the Central regions were similar, but were higher in the North for adults over 18, including women of childbearing age compared against the MPRs of pregnancy and lactation. The Central region had a slightly higher prevalence

93 84 of zinc deficiency for men and women aged than the North, although in both groups the differences between the mean zinc absorbed in the two regions were not statistically significant. The rural regions had substantially higher predicted prevalence of zinc deficiency for all adult groups than did urban areas. Figure 4: Prevalence of Inadequate Zinc Intake, Women, Regional For adult groups, the south had the highest prevalence of zinc deficiency despite having a statistically significantly higher mean dietary zinc intake than the other two regions. This can be explained by the much higher levels of phytate, and subsequently higher phytate to zinc molar ratio, and lower mean absorbed zinc in the South. This is also consistent with the stunting rates which are highest in the South. The higher prevalence in the rural areas was expected based on the lower mean zinc and higher phytate intakes in rural regions. These regional findings for adults underscore the substantial inhibitory effect of dietary phytate on zinc absorption, even at high levels of dietary zinc intake such as in the South. It reaffirms the

94 85 importance of reducing phytate as a means to improve zinc nutrition in high-phytate diets, and suggests that the South has the most to gain from an intervention focused on phytate reduction. By contrast the North, which also has a high deficiency prevalence, had the lowest phytate-to-zinc molar ratio. Here the low lower dietary zinc intake calls for increasing total zinc through biofortification or supplementation, as long as the total phytate levels can be kept low. For physiological groups of children, to which the Miller equations were not applied and the EAR cut-point was used instead, the patterns was different than for adults. For these groups, prevalence of zinc deficiency was highest in the North, followed by the Central, and lowest in the South. The explanation for this lies in the fact that total dietary zinc intakes were lowest in the North and highest in the South. This method uses the EAR and applies a blanket rate of zinc absorption to all regions, and thus did not account for the higher phytate levels in the South. The regional ordering of zinc deficiency rates in children do not align with the regional order of under-5 stunting prevalence rates, nor the regional mean molar ratios. If zinc absorption in children is not heavily dependent on phytate levels as some recent research suggests, then these results could be interpreted as accurate. Further interpretation of the results and consideration of other factors that affect zinc absorption in children, such as environmental enteropathy, are addressed later.

95 Research Question 2 Research Question 2a: What is the expected reduction in the prevalence of zinc deficiency resulting from the adoption of improved household maize processing techniques? Research Finding 2a: Household Processing can substantially reduce the prevalence of inadequate zinc intakes in Malawi. The prevalence fell by over two-thirds in women ages 14 and older. For men prevalence rates fell by nearly half, but remained high. Prevalence rates for children fell 72%, however baseline rates were low and results should be interpreted with caution. Table 14: Population Mean Molar Ratio at Baseline Compared to Various Levels of Coverage of Household Processing Intervention Coverage Level Phytate: Zinc Molar Ratio % Change from Baseline No Intervention 100% Coverage 40% Coverage 20% Coverage 10% Coverage % 16.00% 8.00% 4.00% The simulation of household level maize processing techniques such as soaking, germinating, and fermentation revealed a substantial effect on the zinc nutrition of the population of Malawi. Table 14 displays the mean phytate to zinc molar ratios of the Malawian diet at the national level. Figures are given at baseline and after the simulation of different coverage scenarios of household level processing techniques. At a hypothetical 100% coverage, a reduction in phytate through household maize processing was sufficient to reduce the molar ratio over 40%, to below 15,, the WHO threshold for low zinc bioavailability. Although the gains at lower coverage levels were more modest, coverage levels of 40%, 20%, and 10% were able to reduce the phytate to zinc molar ratio by 16, 8, and 4%, respectively. Nevertheless, this basic analysis reveals that substantial nutritional improvements can be realized from behavior change alone, without altering the quantity or composition of the existing national diet.

96 87 Women Table 15: Prevalence of Zinc Deficiency Risk for Women, Comparison of Household Processing at Various Levels of Coverage (20% coefficient of variation) Females Females Age Age > 18 Females of Childbearing Age (Pregnancy MPR) Females of Childbearing Age (Lactation MPR) Baseline (No Intervention) 34.03% 22.58% 86.49% 96.29% 10% Coverage 31.10% 20.16% 83.78% 95.11% 20% Coverage 27.60% 18.02% 80.76% 93.65% 40% Coverage 22.70% 14.25% 74.39% 90.12% 100% Coverage 11.66% 7.10% 53.15% 74.14% Women s zinc nutrition in Malawi can be substantially improved through an intervention to promote household maize processing techniques to reduce dietary phytate. In women aged 14-18, the simulation of full coverage resulted in a 20% increase in mean absorbed zinc, from 2.16 to 2.60 mg/day. The corresponding percentage of the population with intakes below the MPR for this group fell from 34.04% to 11.66%, a decrease of 65.7%. This substantial reduction suggests that for this vulnerable sub-group, processing alone was sufficient to reduce the prevalence of risk of zinc deficiency below 25%, the IZiNCG threshold for public health priority. At 40% coverage, the prevalence still dropped below the public health priority threshold, while under pessimistic scenarios, the gains realized were smaller (Table 15). For women over 18 the population baseline prevalence of 22.58% was just below the public health priority, but dropped substantially at full coverage to 7.10%, a decrease of over 68%. Partial coverage of the intervention had a more modest impact, reducing the prevalence to 14.25%, 18.02%, and 20.16%. The proportion of women of childbearing age who failed to meet the zinc requirements of pregnancy was reduced by the maize processing simulation as well. The simulation found that at full coverage, mean absorbed zinc of women of childbearing age increased from 2.2 to 2.64 mg/day,

97 88 very near the physiological requirement for pregnancy of 2.68mg/day. This indicates approximately half (53.15%) of women of childbearing age would meet the requirements of pregnancy if phytate were reduced as predicted. The portion of women of childbearing age below the requirement for lactation, however, remained quite high at 74.14% even at full coverage. For both physiological states, the proportion below the requirement remained quite high at lower levels of coverage: 75-84% for pregnancy, and 90-95% for lactation. In an ideal scenario, phytate reduction could reduce the proportion of women failing to meet the zinc requirement for pregnancy to nearly 50%. Nevertheless, in more realistic coverage scenarios, the proportion remained high, and it remained high in all scenarios for breastfeeding women due to their high zinc requirements. Additional targeted interventions, such as zinc supplementation, are likely to be necessary for pregnant and lactating mothers based on this analysis. Men Table 16: Prevalence of Zinc Deficiency Risk for Men, Comparison of Household Processing at Various Levels of Coverage (20% coefficient of variation) Males Age Males Age > 18 Baseline (No Intervention) 71.57% 78.44% 10% Coverage 67.64% 74.92% 20% Coverage 64.01% 70.99% 40% Coverage 54.81% 63.71% 100% Coverage 33.87% 41.49% After simulation of full coverage, the proportion of men age at risk of zinc deficiency fell from 71.57% to 33.87%, a decrease of over 50%, and the mean zinc absorbed increased from 2.26 to 2.75 mg/day. A similar improvement was observed for men over 18, where processing reduced national prevalence by 47.11%, from 78.44% down to 41.49% and increased absorbed zinc from 2.32 to 2.81 mg/day. At simulations of partial coverage for 40%, 20% and 10%, the proportion of men at risk of deficiency was reduced to 63.71%, 70.99%, and 74.82%, respectively. At lower levels of coverage, the proportion of males aged at risk ranged from 55-68%.

98 89 One important implication is that at full coverage, phytate reduction was able to increase the mean zinc absorbed for men in both groups to a level greater than the MPR. In other words, prevalence dropped below 50% for both groups in an ideal, full coverage scenario. Nevertheless the prevalence rates for men remained higher than the 25% public health priority, and although the documented risks to men of zinc malnutrition are not necessarily as great as to children and women of childbearing age, the figures reflect the persistence of the problem of zinc deficiency in Malawi. Children Table 17: Prevalence of Zinc Deficiency Risk for Children Age 9-13, Comparison of Household Processing at Various Levels of Coverage (20% coefficient of variation) Children age 9-13 years Baseline (No Intervention) 9.38% 10% Coverage 8.22% 20% Coverage 7.28% 40% Coverage 5.65% 100% Coverage 2.65% As reviewed, the Miller equations to predict zinc absorption based on zinc and phytate intake are not valid for infants and young children because they are based on studies of adults. As such, the predicted zinc absorption for young children is assumed to be a blanket rate, and not to vary based on individual phytate intake. The Miller equations were applied to children aged 5-9 years and 9-13 years. However for the former group, even the baseline prevalence was so low (<1%) that any reduction seen through simulation could not be meaningfully interpreted. Therefore the only group for which the simulation to reduce phytate was conducted was children aged 9-13 years. After the simulation of household processing at 100% coverage, the proportion at risk of inadequate zinc decreased over 70%, from 9.38% to 2.65%. At 40%, 20%, and 10%, the prevalence was reduced to 5.65%, 7.28%, and 8.22%, respectively. The simulation reveals that household level processing to reduce phytate can reduce the prevalence of zinc deficiency in older

99 children to a very low level, however these results should be interpreted with caution because the baseline prevalence was so low at the start. 90 Research Question 2b: How do these changes vary across regions and between urban and rural residence? Research Finding 2b: At the regional level, the South stands to benefit the most from household food processing. At both full coverage and at 40% coverage, household processing can reduce the proportion of females aged at risk below 25% across all regions. For all physiological groups, the gains from household processing were greater in rural areas than in urban areas. Regional Comparison of the Effect of Processing on Dietary Composition Table 18: Molar ratios at various levels of intervention coverage, along with the percentage change from baseline. No Interventi on 100% Coverage 40% Coverage 20% Coverage 10% Coverage P:Z Molar Pop. Ratio % Change from Baseline 40.68% 16.00% 8.00% 4.00% P:Z Molar North Ratio % Change from Baseline 34.59% 13.99% 7.21% 3.17% P:Z Molar Central Ratio % Change from Baseline 40.60% 15.77% 8.13% 4.19% P:Z Molar South Ratio % Change from Baseline 41.43% 16.51% 7.99% 3.97% P:Z Molar Urban Ratio % Change from Baseline 35.77% 12.85% 7.10% 3.50% P:Z Molar Rural Ratio % Change from Baseline 41.43% 16.52% 8.17% 4.09% + P:Z Molar Ratio: Phytate-to-Zinc Molar Ratio

100 91 A regional comparison of the change in phytate to zinc molar ratios shows that household maize processing had the greatest effect on diet composition in the South. Table 18 shows the molar ratios resulting from the simulation of various levels of coverage, as well as the percent changes from baseline. At full coverage, the relative reduction in molar ratio was greatest in the South, where the ratio was reduced by 41.43%. Still, the South retained the highest overall molar ratio after simulation. This reduction was closely followed by the Central region, which saw a reduction of 40.6%. Gains in the North were more modest though still substantial, at a 34.6% reduction. The North had the lowest average molar ratio both before and after processing. Both urban and rural areas saw substantially reduced molar ratios, 35.77% and 41.43%, respectively, with the gain greater in rural areas. The improvement in the dietary makeup was in line with the expected changes from the baseline analysis in terms of maize consumption and total dietary phytate. Partial coverage of the intervention resulted in smaller reductions in the molar ratio; however partial coverage still represents an opportunity to improve the zinc nutrition of Malawian diets in certain regions. At 40% household coverage, household processing techniques can reduce the mean molar ratio by 16.5% in both the South and for rural households generally. At lower levels of coverage (20% and 10%), rural areas still saw greater improvement than urban areas. At the two lowest levels of coverage however, the percent reduction in the South was no longer greater than the in Central region. Regional Comparison of Reductions in Zinc Deficiency Prevalence through Household Maize Processing Disaggregation of the effect of processing at the regional level shows that the impact aligns with the patterns in dietary consumption seen at baseline. The gains from processing, measured as a reduction in the population at risk of inadequate zinc intakes, were greater in the South and Central

101 regions, and lowest in the North. In addition, the regional differences were more important than the urban-rural distinction, which has implications for programming. 92 The findings are in line with the baseline analysis that the phytate-to-zinc molar ratios were highest in the South, followed by the Central region. In the North, phytate is less of a constraint on zinc absorption than in the other regions, which are also more dependent on maize. Thus the potential gains from reducing phytate are lower in the North, which may benefit more from an increase in dietary zinc, rather a reduction in phytate. These patterns are seen across the various physiological groups analyzed. For women age 14-18, household processing at both full coverage and 40% coverage reduced the prevalence of zinc deficiency below the 25% public health priority threshold for all regional subgroups (Figure 5). The gains for women followed a pattern of greater gains from processing in the Central and South regions than in the North at each level of coverage. In the South at full coverage, processing reduced the proportion of women at risk by 68% and the proportion of women over 18 at risk by 71.15%. In the South, the mean zinc absorbed by women increased by about 23%, while mean zinc absorbed in the North increased by only 14%. In the Central, the prevalence as reduced 64.74% and 67.16% for women and over 18, respectively. In the North the prevalence was reduced by a lower, but still substantial degree: 56.19% and 53.35% for the two female physiological groups. The same results held for the proportion of women of childbearing age who fail to meet the zinc requirements of pregnancy and lactation. At baseline, prevalence was highest for women in the South, followed by the North and closely by the Central region. At 40% coverage, the prevalence rates between the North and South were very similar, while at full coverage, the South obtained a lower prevalence rate of zinc deficiency risk than the north.

102 Table 19: Regional Prevalence Rates of Zinc Deficiency Risk Resulting from Household Level Maize Processing at Various Levels of Coverage for Women MPR GROUP Pop. North Central South Urban Rural Baseline Mean P:Z Ratio % Coverage Mean P:Z Ratio % Coverage Mean P:Z Ratio % Coverage Mean P:Z Ratio % Coverage Mean P:Z Ratio yrs Baseline (fem.) 34.03% 30.29% 31.20% 38.17% 23.84% 36.41% 10% Coverage 31.10% 28.55% 28.39% 34.90% 22.33% 33.13% 20% Coverage 27.60% 26.45% 25.78% 30.02% 19.97% 29.38% 40% Coverage 22.70% 22.61% 20.93% 24.94% 18.01% 23.75% >18 yrs (fem.) Women of CBA Pregnanc y MPR Pregnanc y MPR Pregnanc y MPR Pregnanc y MPR Lactation MPR Lactation MPR Lactation MPR Lactation MPR Lactation MPR 100% Coverage 11.66% 14.13% 11.00% 12.15% 9.33% 12.19% Baseline 22.58% 21.32% 19.79% 26.07% 14.24% 24.60% 10% Coverage 20.16% 19.73% 17.69% 23.14% 12.92% 21.90% 20% Coverage 18.02% 18.09% 15.83% 20.61% 11.74% 19.53% 40% Coverage 14.25% 15.13% 12.77% 15.85% 10.04% 15.22% 100% Coverage 7.10% 9.34% 6.50% 7.52% 5.59% 7.43% Baseline 10% Coverage 20% Coverage 40% Coverage 100% Coverage Baseline 10% Coverage 20% Coverage 40% Coverage 100% Coverage 86.49% 85.32% 83.47% 89.56% 74.80% 88.57% 83.78% 83.42% 80.59% 87.00% 72.13% 85.95% 80.76% 80.99% 77.56% 83.99% 69.01% 83.03% 74.39% 76.41% 71.30% 77.39% 64.66% 76.39% 53.15% 61.45% 50.77% 54.68% 46.40% 54.65% 96.29% 95.80% 94.96% 97.49% 90.37% 97.12% 95.11% 94.94% 93.56% 96.50% 88.72% 96.06% 93.65% 93.77% 91.95% 95.20% 86.68% 94.76% 90.12% 91.31% 88.19% 91.86% 83.59% 91.30% 74.14% 81.15% 71.94% 75.51% 67.70% 75.48% 93

103 94 Figure 5: Estimated Prevalence of Inadequate Zinc Intake for Females Age 14-18, at baseline and after Household Maize Processing Intervention at various levels of coverage. Coefficient of variation of absorbed zinc is assumed to be 20%. Figures above the bars represent the percent reduction in prevalence from baseline. At baseline, men aged had lowest mean absorbed zinc in the South, followed by the Central then North, although only the South was statistically significantly different than the other regions. After the simulation of full coverage, however the mean absorbed zinc in the Central region (2.78mg/day) was higher, and significantly different, than the South (2.71mg/day) and North (2.72mg/day), which were not significantly different from each other. Household processing eliminated the initially significance difference in zinc nutrition between the North and South for this age group. For men over age 18, the relative improvement in the South compared to the North was even more apparent. The mean absorbed zinc was lower in the South than the North at baseline (2.26 vs mg/day), yet after processing, the mean absorbed zinc was higher in the South than North (2.78 vs mg/day). Both differences were statistically significant.