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Novel staple food-based strategies to improve micronutrient status for better health and development in sub-Saharan Africa

Final Report Summary - INSTAPA (Novel staple food-based strategies to improve micronutrient status for better health and development in sub-Saharan Africa)

Executive Summary:
INSTAPA (www.instapa.org) aimed to identify novel staple food-based strategies to improve micronutrient malnutrition for better health and development of women and young children in sub-Saharan Africa. The project focused on the improvement of millet-, sorghum-, maize-, and cassava-based foods through biofortification (WP2), fortification (WP3) and post-harvest processing (WP4), to safely prevent deficiencies of iron, zinc and vitamin A and to improve immune function (WP5) and cognitive development (WP6). Through capacity building, information sharing and strengthening scientific and technological excellence in staple food-based approaches in Africa and Europe (WP7), the project sought to significantly contribute to the improvement of the dietary quality of young children and their mothers living in resource poor and malaria endemic areas in sub-Saharan Africa. The 5½ -year project started in June 2008 with research scientists from universities and research institutes from Europe (The Netherlands, United Kingdom, France and Switzerland) and Africa (South Africa, Kenya, Benin, Nigeria, Burkina Faso and Mali) coordinated by Dr. Inge D. Brouwer, Wageningen University (inge.brouwer@wur.nl). Fieldwork for INSTAPA was carried out in Burkina Faso, Mali, Benin and Kenya.
Main results indicate that INSTAPA succeeded in producing varieties of pro-vitamin A biofortified or yellow cassava with sufficient beta- carotene levels that are generally well accepted. Production and consumption of yellow instead of white cassava would offer an alternative option for reducing vitamin A deficiency in sub-Saharan Africa. Iron biofortification of pearl millet is a promising approach as consumption would double the amount of iron absorbed. Biofortification via soil fertilization with zinc appears to be as good an intervention strategy to combat zinc deficiency as the conventional zinc fortification of foods. Genetic diversity of iron and zinc concentrations of sorghum exists but at present the potential contribution of sorghum varieties to iron and zinc intake is not sufficient to solve iron and zinc deficiency. The use of NaFeEDTA seems to be the most feasible and promising approach increasing iron bioavailability from iron-fortified sorghum foods containing both phytic acid and inhibitory polyphenols. In remote areas with limited access to conventionally fortified foods, in-home fortification with millet-based complementary foods using enhancers, such as phytase and ascorbic acid, is a promising approach to provide additional iron. Concerning zinc fortification of cereal staples, zinc sulphate is a slightly better absorbed fortificant than zinc oxide, while EDTA is a modest enhancer of zinc absorption. But adequate absorption can be obtained with zinc oxide making cost and sensory properties the most important criteria for selection. Phytase enzyme added before consumption can significantly increase Zn bioavailability in adults and children. Its potential addition to micronutrient powders or lipid-based nutrient supplements for home fortification may be of important public health significance. Due to the low mineral contents and high contents of factors inhibiting absorption in sorghum and millet, and to the observed effects of decortication (co-elimination of desired and undesired compounds), the optimisation of the contents in bioavailable iron and zinc in millet or sorghum-based dishes through improvement of processing is probably not sufficient. Formulation of optimized accompanying leafy vegetable sauces is potentially interesting, but the total quantity of iron absorbed from these improved sauces was not higher than that of traditional sauces. Further measures such as adding iron absorption enhancers to the sauces, decreasing the concentration of inhibiting phenolic compounds in the sauces need to be investigated. Very importantly, studies showed that asymptomatic malaria parasitemia decreases dietary iron absorption through low-grade inflammation. This may contribute to iron deficiency or iron deficiency anemia or may blunt efficacy for fortification programmes in malaria-endemic areas. Antenatal iron supplementation leads to large improvements of birth weight, fetal growth and infant iron stores, with potentially immense benefits for infant survival and health that should outweigh any possible concerns about risks of malaria. Scaling up universal iron supplementation, even in the presence of universal iron fortification of staple-foods, in pregnancy in developing countries is likely to generate major public health gains. Addition of micronutrient powders to maize flour is beneficial to hemoglobine levels of young children and improved health status and behaviour, the latter detectable after 2 years of age.
Project Context and Objectives:
Poor nutritional status and limited financial resources often compromise individual welfare in the developing world. In combination with a commonly high concurrent disease load, a self-perpetuating cycle of poverty, malnutrition and mortality arises. Adequate nutrition is a human right that underpins progress towards most of the Millennium Development Goals (MDG) as formulated under the banner of the United Nations (2000) to be reached in 2015, such as reducing child mortality, improving maternal health and decreasing the burden of malaria, HIV/AIDS and other diseases. It will also affect the achievement of future Sustainable Development Goals when not addressed properly. Although the proportion of undernourished people in developing countries reduced, the reduction is modest and still leaves more than 800 million people undernourished. Malnutrition remains a pervasive and damaging problem of public health concern in many low and middle income countries. To date it is estimated that over 165 million children under five years of age are stunted and about 52 million children are too thin (wasted) requiring special treatment. In the past 15 years the rate of malnutrition has reduced, however, the improvement is stagnating especially in sub-Saharan Africa where the absolute number of undernourished people continues to rise. Undernutrition remains the dominant contributor to the disease burden in this region being responsible for over 45% of total deaths of children younger than 5 years. Thus, improving nutritional status especially in sub-Saharan Africa is critical to break the cycle of malnutrition, poverty and mortality.

Apart from energy and protein malnutrition, there is rising concern about the nutritional quality of diets in terms of micronutrient adequacy in developing countries. Sheer hunger, caused by lack of calories and proteins, is mostly found in areas of emergencies caused by e.g. civil war or extreme drought. Hidden hunger, which is the lack of micronutrients, affects a far larger number of the world’s population. Over a quarter of the world’s population is anaemic, including 293 million children (47%) younger than 5 years and 486 million (30%) non-pregnant women. In sub-Saharan Africa, the prevalence of anaemia in pregnant women was reported to be 68% in Burkina Faso and in the range of 47-63% in Mali. Two surveys in Benin suggested anaemia prevalence rates of 82% among children aged 6-59 months and of 60% in children 36 to 60 months. A major cause of anemia is iron deficiency having adverse health effects on pregnancy outcome, infant growth, cognitive performance, psychomotor development, immune status and work capacity, with substantial economic costs related to impaired school performance and decreased productivity. For pregnant women, anaemia contributes to over 20% of all maternal deaths. Even mild-to-moderate iron deficiency without anaemia may reduce work capacity and resistance to fatigue and impair cognition. The risk of deficiency is highest when iron needs are proportionately greater than energy needs, making infants and young children particularly vulnerable. In many developing countries, use of plant-based weaning foods contributes to a high prevalence of anaemia in young children.
Because iron and zinc are found in many of the same foods, high rates of iron deficiency in sub-Saharan Africa suggest widespread occurrence of zinc deficiency in the same populations. Zinc is required by ~50 enzymes in the body, and many metabolic functions are affected by zinc deficiency, including physical growth, immune competence, reproductive function and neural development. Several supplementation trials among high-risk young children have shown that increased zinc intake reduces the burden of diarrhoea and acute lower respiratory infections. Estimates suggest that 17% of the world population is at risk of zinc deficiency, on the basis of an analysis of national diets. Especially in sub-Saharan Africa young children have inadequate dietary intake of bioavailable zinc, leading to zinc deficiency as assessed by plasma or serum zinc concentrations.

Vitamin A deficiency affects an estimated 90 million preschool-age children (33%) and 19 million pregnant women (15%). Approximately every third child with vitamin A deficiency lives in sub-Saharan Africa. Health consequences of vitamin A deficiency comprise mild to severe systemic effects on innate and acquired immunity, increased burden of infectious morbidity, xerophthalmia, blindness, and increased mortality. Although clinical signs of vitamin A deficiency show a downward trend, even subclinical vitamin A deficiency is associated with excess maternal and infant mortality. Sub-Saharan African countries are among those with the highest vitamin A deficiency prevalence, often in excess of 50%.

Major causes of micronutrient deficiencies comprise low intake due to food scarcity, unavailability of micronutrient-rich foods, poverty, or poor dietary habits; high demands that arise from growth spurts during infancy and adolescence, pregnancy, or increased blood loss as occurs with pronounced menstruation or infestation from parasites; and low bioavailability of micronutrients. Africa's traditional weaning foods are watery gruels based on boiled cereals. Such diets are rich in substances that inhibit iron and zinc absorption such as phytic acid and polyphenols, while poor in ascorbic acid and meat that enhance iron bioavailability. The intestinal absorption of provitamin A ingested from plant foods to vitamin A is much less than previously thought. This is particularly the case for dark-green leafy vegetables, which contain ß-carotene trapped in chloroplast membranes, but bioavailability may be higher for ß-carotene from tubers that do not contain chloroplasts. An expert group recently concluded that it is virtually impossible to correct widespread vitamin A deficiency by conventional plant-based diets alone in developing countries. Foods that are good sources of bioavailable iron, zinc and vitamin A, such as animal products, are generally not available due to high costs or limited supply, especially to poorer populations in the developing world.

At the World Summit for Children in 1990, elimination of deficiencies of vitamin A, iron, and iodine were placed high on the agenda. Zinc was added to the list in the Third Report on the World Nutrition Situation. The urgency of addressing micronutrient malnutrition has substantially been boosted by a series of global consensus articles published in the Lancet in 2008 and 2013, summarizing the best evidence for addressing maternal and child undernutrition. Since then, the global Scaling Up Nutrition (SUN) movement is targeting national governments to create awareness on undernutrition, and nutrition is now being taken up in national policies more than ever. During the last decade, the value of adequate nutrient status has been underlined by the Copenhagen Consensus, in which world top economists have stated now three times in a row (2004, 2008 and 2012) that the provision of micronutrients to deficient populations is the most efficient investment among all urgent global challenges of our time.

Supplementation strategies have been widely used to provide iron and folic acid to pregnant women, and vitamin A to infants, young children and postpartum to women. However, supplementation usually requires the procurement and purchase of micronutrients in a relatively expensive pre-packaged form, an effective distribution system and a high degree of consumer compliance. Lack of supplies, low coverage and poor compliance are consistently reported as being the main barriers to sustainable success. Safety is also a concern. For example, vitamin A is toxic when ingested in large quantities, this in contrast to dietary provitamin A. Moreover, the recent restrictions on supplementation with iron in malaria-endemic areas by WHO has brought international organizations that strive to alleviate iron deficiency anaemia in such regions in an impasse. Food-based approaches are the only way to overcome this. As most of the resource poor people are depending on staple foods for most of their nutrients, improving the nutrient density of these staple foods will contribute in a sustainable way to a reduction of micronutrient deficiencies.

Fortification and biofortification offer the potential of reducing micronutrient malnutrition as part of a food-based approach when existing food supplies or supplementation strategies fail to provide adequate levels of the respective nutrients in the diet. Dietary approaches such as improved processing or food-to-food fortification have the potential to improve micronutrient supply or bioavailability, and could synergistically enhance the efficacy of (bio)fortification. The research not only aims to improve the nutritional quality of the diets especially of children in low-income countries, but also to measure the effect of this improvement on functional outcomes of immunity, infection and cognitive development. Such data is urgently needed in order to help to build a common ground for inclusion of nutrition in the agendas of policy makers.

INSTAPA aimed to identify novel staple food-based strategies to improve micronutrient malnutrition for better health and development of women and young children in sub-Saharan Africa. It focused on the improvement of millet-, sorghum-, maize-, and cassava-based foods. Millet, sorghum, maize and cassava are major staple foods in sub-Saharan Africa. Despite being the world's fifth and sixth major crops (after rice, wheat, maize, and potatoes), sorghum and millet have not yet been developed as major foods for urban markets in Africa and therefore remain crops of the small cultivator. Consumption levels and micronutrient bioavailability from these crops remain largely unknown. The development of a sorghum- or millet-based food-processing industry would do much to offset Africa's shift in demand towards imported rice and wheat. Cassava (Manihot esculenta) is widely grown in the tropics and is a low-cost source of dietary energy and protein. However, the nutritional quality of traditional cassava varieties is poor. Introducing provitamin A-rich yellow cassava from Latin-America in Africa could help combat widespread vitamin A deficiency especially in the most resource-limited regions. Maize is the most important staple food in Eastern and Southern Africa and is expanding rapidly into traditional sorghum growing areas. Its native high phytate content inhibits the absorption of iron and zinc. Yellow maize, which is high in carotenoids, has marginal significance as a seasonal snack in Africa. More widespread use of yellow maize could help improve vitamin A status of populations relying heavily on this staple food.
INSTAPA investigated the potential of biofortification, fortification, and post-harvest processing, to safely prevent deficiencies of iron, zinc and vitamin A and to improve immune function and cognitive development. Specifically, the objectives were:
• To evaluate the genetic potential of cassava, maize, millet and sorghum for increased content of provitamin A, iron and zinc and for reduced content of antinutrients;
• To determine the efficacy of fortified and biofortified cereal-based foods on nutritional status and health;
• To develop improved (traditional) processing methods of millet, sorghum and maize enhancing iron, zinc and vitamin A bioavailability and/or reducing inhibitory effects of anti-nutrient factors for improvement of micronutrient status of young children and women;
• To determine the safety and efficacy of iron-fortified maize to alleviate anaemia in malaria-endemic areas in sub-Saharan Africa;
• To determine the effect of maize-based complementary foods fortified with iron and zinc on cognitive and psychomotor development of young children.
Through capacity building, information sharing and strengthening scientific and technological excellence in staple food-based approaches in Africa and Europe, INSTAPA sought to significantly contribute to the improvement of the dietary quality of young children and their mothers living in resource poor and malaria endemic areas in sub-Saharan Africa. It aimed to establish an international leading role of the consortium in the fields of biofortification, fortification and processing to increase supply of bioavailable micronutrients from African staple crops by bringing together scientific and technological excellence. Capacity building in knowledge and skills to solve micronutrient deficiencies in sub-Saharan Africa and Europe was aimed to be strengthened through training of young scientists and professionals from Africa and Europe embedded in a coherent set of inter-related activities. INSTAPA aimed to contribute to enabling African populations to define and choose diets for optimal health for their children through sharing knowledge and expertise to maximise the speeds of impact of the advances in solving micronutrient malnutrition. Novel knowledge and technologies was aimed to be made available to strengthen the competitiveness of local SME (farmers, millers, and food processors) as well as African and European food industry targeted at evidence-based production of healthier complementary foods for African children.

The 5-year project started in June 2008 with research scientists from Europe and Africa, coordinated by Dr. Inge Brouwer, Wageningen University. Activities were divided across 7 complementary Work Packages to cover key aspects of the INSTAPA project, being Biofortication (WP2), Fortification (WP3), Post-harvest processing (WP4), Safety of food-based interventions (WP5), Cognitive development (WP6) and Capacity building and dissemination (WP7), coordinated and management by WP1 (www.instapa.org).
Project Results:
As most people depend on food for their (micro) nutrients, knowledge of the existing dietary patterns and nutrient intake is essential for the development of food-based approaches to alleviate micronutrient deficiencies. In INSTAPA several food consumption studies have been carried out in Benin, Burkina Faso, Mali and Kenya. In Mali, a food consumption study among young children and women in child bearing age showed that only 2 out of 106 children had an adequate energy intake, while none of the children nor women had sufficient iron and zinc intakes. The main dietary sources of iron and zinc did not differ between children and their mothers. The main iron sources were sorghum (30%), legumes (12%), vegetables (10%), green leafy vegetables (LV; 10%) and millet (12%), whilst the main sources of zinc were sorghum (32%), rice (17%), millet (16%) and legumes (9%). Meat accounted for only 1.6% of the iron intake. In Kenya, a food consumption survey among schoolchildren showed that in general the children had a low energy intake. Although iron intake seemed to be sufficient, 100% of the children showed a vitamin A (VA) intake below their average estimated requirement. In addition, more than 15% of the children had an insufficient zinc intake. In Benin, red palm oil, palm nut sauce, various LV sauces, mango and egg were the main VA-rich foods consumed by young children. Eggs were the only VA-rich foods of animal origin, but eggs were consumed only by 1.4% of the children surveyed. None of the children ate liver or butter. In Burkina Faso, among children 6 - 35 months iron and zinc intakes were far below the recommended intakes. High fibre and phytate intakes were also observed. More than 90% of the young children consumed millet, sorghum or maize, while 55% and 67% of the children consumed meat and fish, however in small amounts.

The food consumption surveys indicated that in the African countries surveyed, vitamin A, iron and zinc intakes were low. Most of the diets were based on staple foods, providing most of the micronutrients. Based on the food consumption surveys, INSTAPA identified and characterised the processing methods of the main traditional foods consumed, mainly in Benin and Burkina Faso. The traditional recipes of millet-, sorghum-, and maize-based dishes and the accompanying sauces were described including the uses of the dishes, the processing steps well as the type and amount of ingredients used. The nutritional composition of these main dishes and accompanying sauces related to macronutrients and iron, zinc, carotenoid, retinol and myo-isotol hexaphosphate (IP6) were determined. The recipes and nutritional composition was published in a booklet in both English and French, and this formed the basis for further elaboration on the potential for improving the iron, zinc and vitamin A density of these traditional dishes and accompanying sauces.

1 Evaluation of the genetic potential of cassava, maize, millet and sorghum for increased content of provitamin A, iron and zinc and for reduced content of antinutrients

Biofortification, the process of breeding nutrients into food crops, provides a sustainable, long-term strategy for delivering micronutrients to rural populations in developing countries. For biofortification to be successful, a first question to be addressed is whether breeding can increase the micronutrient density in food staples to target levels that will make a measurable and significant impact on nutritional status. In addition to genetic factors, also environmental factors during the growth and grain filling period determine iron (Fe), zinc (Zn) and phytate concentration in the grain. In INSTAPA biofortification efforts were focused on sorghum, millet (related to iron and zinc) and on cassava (related to vitamin A).

1.1 Sorghum
The INSTAPA work on sorghum aimed to achieve genetic progress in selection of varieties with high iron and zinc that are acceptable for cultivation and consumption by farmers in West Africa. The following objectives were addressed:
- To estimate the relative importance of environmental conditions (growing conditions, especially with respect to the extremely low phosphorus availability found in women’s fields) versus genetic effects for determining the status of mineral concentrations in the grain, as well as phytate.
- To estimate the levels of mineral losses due to decortication, using the available tools to women in rural areas, as well as estimating the repeatability of these estimates, and the resultant iron and zinc concentrations available in diets of young children and women of childbearing age.
- To estimate repeatability, genetic and genetic by environmental components of variance of iron, and zinc concentrations under standardized testing conditions. This was be done in view of trait relationships for morphological and agronomic traits and iron and zinc concentrations in sorghum, to assess whether certain grain morphological and growth related traits have a major influence on iron and zinc concentrations.

Studies on the diversity for iron and zinc content in West –African sorghum germplasm, revealed that sorghum is a very diverse species. In INSTAPA 66 different varieties were evaluated for iron and zinc content across two sites with trials conducted under both high and low phosphorus (P) conditions, over a period of 3 years. The results showed that grain grown under poor soil fertility (low P availability) conditions had on average 11% lower Fe and Zn concentrations, than grain cultivated under adequate soil fertility conditions. Genetic differentiation between varieties was highly repeatable, also at low P-availability conditions. We did not detect any interactions between genotypes and soil fertility conditions for both Fe and Zn concentrations, thus selection for high Fe and Zn concentration is possible under any growing condition. The highest Fe and Zn varieties were local varieties from West Africa. Much of the new breeding material actually performed poorer than the traditional varieties for Fe and Zn concentrations. It is thus important to monitor Fe and Zn in newly released varieties, and ensure a minimum standard. The group of varieties with the highest Fe concentration achieved an average of 24 ppm, thus approaching the level required for improved nutritional intake of children, if the whole grain is consumed.
Sorghum grain is traditionally consumed as flour or grits from decorticated grain. The ease of decortication and the overall decortication losses are an important varietal characteristic for sorghum in West Africa. INSTAPA research, conducted in 13 villages across southern Mali, shows that manual decortication lead to grain weight losses of 18 - 35%, depending on the variety. Associated with these losses on grain weight are losses of iron and zinc of 50% on average. The decortication seems to have no effect on bio-availability of iron and zinc, as IP6, part of the phytate in the grain was also lost at about the same rate. Thus the molar ratios for whole grain and decorticated grains were very similar. The consumption of whole grain should thus be advantageous in terms of improving mineral nutrition in rural areas.

INSTAPA also looked for opportunities for increasing production of high iron sorghum varieties by women farmers in southern Mali. Women grow sorghum as an intercrop in their groundnut or cowpea fields. These fields tend to have extremely low soil fertility with lower P availability levels than in the low P research trials performed on station. Women use the sorghum produced by their own for preparing extra meals for young children, as well as for selling and purchasing ingredients for the sauce to accompany the daily meals. The women thus tested some of the highest iron and zinc varieties in their own fields, with and without treatments for improving soil fertility. The results indicated that the women like the highest Fe/Zn variety (Douaje) very much, and are starting to cultivate it on a larger scale. 30% grain yield improvements have been achieved with application of wood ash in the planting holes at the time of sowing.
In conclusion, INSTAPA studies indicated that genetic diversity in iron and zinc concentrations of sorghum exists but at present the potential contribution of sorghum varieties to iron and zinc intake is not sufficient to solve the iron and zinc deficiency. Attempts to identify low phytate varieties have not succeeded. However, caution should be taken to prevent release of new varieties with a lower content of iron and zinc content. As a consequence of INSTAPA, ICRISAT has stated in its documents describing the impact pathways, theory of change and the plans of work for the CGIAR Research Program (CRP) on dry land Cereals, that ICRISAT will only release or recommend for adoption sorghum and millet varieties that have satisfactory (as good or better contents of Fe and Zn than local varieties) levels of Fe and Zn concentration. Through this measure ICRISAT and the other CRP partners plan to ensure that varieties that are more productive will not only improve food security, abut make a contribution to nutrition security.

1.2 Cassava
At start, 56 cassava genotypes, introduced in-vitro, were transported from Nigeria (IITA) to Kenya (KARI) and were first multiplied rapidly in the laboratory to increase planting materials of each genotype and to maintain the germplasm for future use. The 129 introduced as clean clonal materials from in-vitro plantlets were planted directly in the field. Of these, 99 genotypes survived and the growth at nine months after planting was impressive. After harvesting, spectrophotometric and HPLC analysis were undertaken for analysis of carotenoids concentration. On average, the carotenoid concentration was 8.4 ± 5.6 µg/g. A cut-off of 9 µg/g beta-carotene determined by HPLC was used to define promising varieties. 46 promising varieties were replanted of which 43 were harvested and analysed. The average carotenoid concentration was 5.7 ± 2.9 µg/g which was lower than the concentrations found in the previous harvest. This has been attributed to the shorter duration of growth (8 vs. 12 months), and to the different soil/climate conditions. Based on these data, 11 varieties, all with carotenoid concentrations >7 µg/g were selected for final multiplication an replanting to enable supply of 5,000 kg of yellow cassava for the trial to assess efficacy of yellow cassava consumption on vitamin A status of schoolchildren in Kenya (see 2.2).

Retention studies have been performed, measuring carotenoid content in cassava before and after processing and storage. These studies confirmed that boiling of cassava leads to a carotenoid retention of 50% indicating that half of the amount of carotenoids is destructed or evaporated during preparation.

1.3 Acceptability of biofortified staple crops
An important aspect of the potential of biofortified crops to contribute to improvement of micronutrient deficiencies is whether the population finds the crops acceptable, both in terms of sensory aspects as in terms of socio-cultural acceptability.

Sensory evaluation of yellow cassava was carried out in Kenya and Benin. Sensory acceptability was measured by replicated discrimination tests and paired preference tests among 30 children (7–12 years) and 30 caretakers (18 – 45 years) in three primary schools in Kenya. Caretakers and children perceived a significant difference in taste between white and pro-vitamin A rich cassava. Both preferred pro vitamin A rich cassava over white cassava because of its soft texture, sweet taste and attractive colour. The sensory evaluation of biofortified cassava in Djidja township, Benin, indicated that gari and boiled cassava are the two cassava products mostly consumed by the population of this locality. The study showed that the yield of commercial gari produced from beta-carotene biofortified cassava was 15.2% (dry basis) against 17% with the same variety in Ibadan, Nigeria. These yields are relatively low compared to that of gari produced with the local white cassava (27%). Preference tests revealed that beta-carotene biofortified cassava products were preferred over white cassava products. Furthermore, the yellow gari produced in Ibadan was significantly preferred over the yellow gari produced in Djidja, using another processing technology. This suggested the need for transfer of technology into the locality. Biofortified cassava products are accepted by both the children and their mothers

Sensory evaluation of biofortified maize was carried out in Benin to determine the effect of biofortification of maize on taste through triangle tests and preference tests. Results indicated that maize is mainly consumed as dihou, bita or sorun and monbou. Products from yellow maize were more preferred than white maize products and easily identifiable in the sensory test. The yellow maize products consumption was mainly determined by the pleasant taste and the attractive colour of these products. Sensory acceptability of biofortified sorghum was studied in Mali, and results indicate that preference scores were above average and comparable among varieties.

Factors predicting the consumption of biofortified maize and cassava by children were determined in Benin and Kenya using the constructs of the Theory of Planned Behaviour and the Health Believe Model. Results in Kenya concerning the yellow cassava indicated that knowledge about pro-vitamin A rich cassava and it’s relation to health (‘Knowledge’ (b = 0.29 P = 0.01)) was a strong predictor of ‘Health behaviour identity’. Worries related to bitter taste and colour (‘Perceived barriers 1’ (b = 20.21 P = 0.02)) the belief of the caretaker about having control to prepare cassava (‘Control beliefs’ (b = 0.18 P = 0.02)) and activities like information sessions about pro-vitamin A rich cassava and recommendations from health workers (‘Cues to action’ (b = 0.51 P = 0.01)) were the best predictors of intention to consume pro-vitamin A rich cassava. Results in Benin concerning yellow cassava, showed that through health behaviour identity, knowledge and perceived susceptibility predicted intention to consume yellow cassava. Consumption of yellow cassava can be promoted by increasing the intention with special attention paid to increasing knowledge about vitamin A deficiency (VAD) and health benefits of yellow cassava and increasing positive triggers to yellow cassava consumption such as recommendations from influential people and educational campaigns. Concerning yellow maize in Benin, results indicated that mothers have knowledge and a positive attitude towards the consumption of yellow maize and there are few barriers. However, mothers are not yet aware of the risk of their own child to develop vitamin A deficiency and how serious this problem is. This affects the health behaviour identity, which in turn influences the intention to consume yellow maize. For introduction of yellow maize in Benin, consumption could be increased by increasing awareness of the risks of VAD of the child making use of subjective norms such as family members or local authorities.

Overall, all biofortified crops were generally well accepted; factors determining consumption differ per country and should be taken into account when introducing biofortified crops.

2 Efficacy of fortified and biofortified cereal-based foods on nutritional status and health

2.1 Efficacy of daily consumption of provitamin A-biofortified cassava on vitamin A status in children aged 5-13 years in rural Kenya
In order to provide proof of principle of the efficacy of yellow cassava (Manihot esculenta Crantz) on improving vitamin A status of a potential target population, we undertook a randomized controlled trial in Kenyan school children aged 5-13 years. The study was conducted in three primary schools in Kenya with 342 children randomly allocated to three intervention groups: 1) 375 g of white cassava and a placebo capsule 2) 375 g of white cassava and a beta-carotene supplement (1054 µg beta-carotene) 3) 375 g of provitamin A rich cassava and a placebo capsule. Cooked cassava was mashed with salt and 4 g of oil per portion; in case of provitamin A rich cassava providing approximately 50% retinol activity equivalent (RAE) of the age-specific EAR (Estimated Average Requirement). Children received the intervention 6 days per week for 18.5 weeks. Field staff and participants were blinded to supplementation. Before and after intervention, venous blood was drawn for assessment with serum retinol concentrations as the primary endpoint. Complete data were collected for 335 children in the randomized controlled study. Compliance to cassava feeding was on average 100% in all three groups combined and was not different between treatment groups. Our population was marginally vitamin A deficient with a mean serum retinol concentration of 0.80 0.83 and 0.82 µmol/L in the control, yellow cassava and beta-carotene supplement group respectively. Primary analyses showed a treatment effect for serum retinol concentration of 0.04μ mol/L (95%CI: 0.03 0.07) in the yellow cassava group and of 0.04 μmol/L (95%CI: 0.03 0.07) in the beta-carotene supplement group. Subgroup analyses did not show a different intervention effect in children with and without vitamin A deficiency or zinc deficiency. This study showed that provitamin A biofortified cassava improves the vitamin A status of primary school children in Kenya.

2.2 Iron absorption from (bio) fortified sorghum and millet based diets or accompanying nutrient supplements in malaria endemic areas
Iron fortification may be a cost-effective, sustainable, and potentially safe strategy to improve iron intakes and reduce anemia. Disappointingly, most trials of iron fortification in malarial-endemic areas of Africa have been ineffective or have only a limited impact. One reason for the blunted effect could be poor absorption due to chronic inflammation from parasite diseases, such as malaria. Therefore, measuring the effect of asymptomatic parasitemia on host iron absorption and utilization may provide insights into the aetiology of anemia in the tropics. Iron absorption and utilization from an iron-fortified sorghum porridge (3 mg iron) were estimated by using oral and intravenous isotope labels in 23 afebrile Beninese women with a positive malaria smear (asexual P. falciparum parasitemia; >500 parasites/μL blood). The women were studied while infected, treated, and then restudied 10 days after treatment. Iron status, hepcidin, and inflammation indexes were measured before and after treatment. Clearance of asymptomatic malaria parasitemia increased dietary iron absorption (from 10.2% to 17.6%; P < 0.01) but did not affect systemic iron utilization (85.0% compared with 83.1%; NS). Malaria treatment reduced low-grade inflammation, as reflected by decreases in serum ferritin, C-reactive protein and interleukin-6, -8, -10 (P < 0.05); this was accompanied by a reduction in serum hepcidin of approximately 50%, from 2.7 to 1.4 nmol/L (P < 0.01). Treatment decreased serum erythropoietin and growth differentiation factor 15 (P < 0.05). The study shows that asymptomatic malaria parasitemia decreases dietary iron absorption but does not influence systemic iron utilization. The negative effect is mediated through low-grade inflammation and it may contribute to iron deficiency (ID) and iron deficiency anaemia (IDA) or may blunt efficacy of fortification programmes in malaria-endemic areas. Further research is needed to investigate whether asymptomatic malaria has a long-term effect on iron absorption from fortified foods, which then would require means to improve iron bioavailability or the adjustment of iron concentrations in fortified sorghum and millet foods in malaria endemic areas.

In contrast to the majority of cereals, millets and particular sorghum can contain considerable amounts of polyphenols. In addition to phytate, polyphenols (PPs) might contribute to low iron bioavailability from sorghum-based foods. To investigate the inhibiting effects of sorghum PPs on iron absorption and the potential enhancing effect of ascorbic acid (AA), NaFeEDTA and the polyphenol oxidase enzyme laccase, we performed three iron absorption studies in 50 young women consuming dephytinized iron-fortified test meals based on white and brown sorghum varieties with different PPs concentrations. Iron absorption was measured as erythrocyte incorporation of stable iron isotopes. In study 1, iron absorption from meals with 17 mg PPs (8.5%) was higher than from meals with 73 mg PPs (3.2%) and 167 mg PPs (2.7%; P < 0.001). Absorption from meals containing 73 and 167 mg PPs did not differ (P = 0.9). In study 2 iron absoption from NaFeEDTA fortified meals (167 mg PPs) was higher than from the same meals fortified with FeSO4 (4.6% vs. 2.7%; P < 0.001) but still lower than from FeSO4-fortified meals with 17 mg PPs (10.7%; P < 0.001). In study 3, laccase treatment decreased PPs from 167 to 42 mg (4.8%) but did not improve absorption compared with meals with 167 mg PPs (4.6%; P = 0.4) whereas adding AA increased absorption to 13.6% (P < 0.001). These findings suggest that PPs from brown sorghum contribute to low iron bioavailability from sorghum foods and that AA and to lesser extent NaFeEDTA, but not laccase, have the potential to overcome PP inhibition and improve iron absorption from sorghum foods. However, application of ascorbic acid in iron-fortified sorghum foods would be challenging due to temperature and oxidation sensitivity of ascorbic acid. Therefore, the use of NaFeEDTA seems to be the most feasible and promising approach increasing iron bioavailability from iron-fortified sorghum foods containing both phytic acid and inhibitory PPs.

Based on the food consumption studies carried out in Benin and Burkina Faso, it became obvious that sorghum was not centrally milled. This hampered the potential of post-harvest fortification to reduce iron deficiency. Where there is no central milling, home fortification with a lipid-based complementary fortified food is promising to improve bioavailable iron and energy intake of young children in developing countries. To optimize iron bioavailability from an lipid-based nutrient supplement (LNS) termed complementary food fortificant (CFF), 3 stable isotope studies were conducted in 52 young Beninese children. Test meals consisted of millet porridge mixed with CFF and ascorbic acid (AA). Study 1 compared iron absorption from FeSO4-fortifed meals with meals fortified with a mixture of FeSO4 and NaFeEDTA. Study 2 compared iron absorption from FeSO4-fortifed meals without or with extra AA. Study 3 compared iron absorption from FeSO4-fortified meals with meals containing phytase added prior to consumption, once without or once with extra AA. Iron absorption was measured as erythrocyte incorporation of stable isotopes. In study 1, iron absorption from FeSO4 (8.4%) was higher than from the mixture of NaFeEDTA and FeSO4 (5.9%; P < 0.05). In study 2, the extra AA increased absorption (11.6%) compared with the standard AA concentration (7.3%; P < 0.001). In study 3, absorption from meals containing phytase without or with extra AA (15.8 and 19.9%, respectively) increased compared with meals without phytase (8.0%; P < 0.001). The addition of extra AA to meals containing phytase increased absorption compared with the test meals containing phytase without extra AA (P < 0.05). These findings suggest that phytase and AA, and especially a combination of the two, but not a mixture of FeSO4 and NaFeEDTA would be useful strategies to increase iron bioavailability from a CFF mixed with cereal porridge. The CFF developed INSTAPA is, similarly to previous LNSs, in form of a paste and contains a mixture of micro- and macronutrients which should be added as a fortificant to cereal-based porridges commonly consumed by children in developing countries. The CFF was designed as a preventive food to treat mild to moderate malnutrition. The CFF is based on ingredients that are locally available in developing countries e.g. soybean flour. Compared with previous LNSs, the soybean flour replaced the milk powder. It is cheaper than milk powder, but on the down side introduces more phytate in the CFF. In such a type of CFF containing considerable amounts of phytate, the results obtained in this thesis support the use of microbial phytase active at gastric pH in combination with increased AA concentrations to improve iron bioavailability. The best way to provide the phytase with the CFF would be to blend the phytase together with the other ingredients of the CFF. Such an approach requires research on the storage stability of phytase in a lipid-dense food matrix. Microbial phytase from Aspergillus niger has been approved by the Joint FAO/WHO Expert Committee on Food Additives and an ADI "not specified" was allocated to applications such as LNSs.

Iron biofortification of pearl millet (Pennisetum glaucum) is a promising approach to combat iron deficiency (ID) in the millet-consuming communities of developing countries. To evaluate the potential of iron-biofortified millet to provide additional bioavailable iron compared with regular millet and post-harvest iron-fortified millet, an iron absorption study was conducted in 20 Beninese women with marginal iron status. Composite test meals consisting of millet paste based on regular-iron, iron-biofortified, or post-harvest iron-fortified pearl millet flour accompanied by a leafy vegetable sauce or an okra sauce were fed as multiple meals for 5 days. Iron absorption was measured as erythrocyte incorporation of stable iron isotopes. Fractional iron absorption from test meals based on regular-iron millet (7.5%) did not differ from iron-biofortified millet meals (7.5%; P = 1.0) resulting in a higher quantity of total iron absorbed from the meals based on iron-biofortified millet (1125 μg vs. 527 μg; P < 0.0001). Fractional iron absorption from post-harvest iron-fortified millet meals (10.4%) was higher than from regular-iron and iron-biofortified millet meals (P < 0.05 and P < 0.01 respectively), resulting in a higher quantity of total iron absorbed from the post-harvest iron-fortified millet meals (1500 μg; P < 0.0001 and P < 0.05 respectively). Results indicate that consumption of iron-biofortified millet would double the amount of iron absorbed and, although fractional absorption of iron from biofortification is less than that from fortification, iron-biofortified millet should be highly effective in combatting ID in millet-consuming populations. These results indicate that iron-biofortified and post-harvest iron-fortified pearl millet have the potential to provide additional bioavailable iron in the diets of pearl millet consuming communities compared with regular-iron pearl millet. However, postharvest fortification of millet is challenging due to the lack of centralized milling and due to the difficulties when fortifying millet on a community-level. Therefore, in communities with limited access to conventionally fortified foods, iron-biofortified millet seems to be the most promising approach to provide additional bioavailable iron. Studies investigating the efficacy and effectiveness of iron-biofortified crops are needed to prove that the additional bioavailable iron is sufficient to improve iron status in the targeted population.

Putting all the findings together, the investigations highlight that improving iron nutrition from sorghum and millet diets in malaria endemic areas is challenging because the negative influences of PA, PPs and malaria on iron absorption have to be considered simultaneously. Nevertheless, the thesis demonstrates that in remote areas with limited access to conventionally fortified foods, in-home fortification of millet-based complementary foods using enhancers, such as phytase and AA, or iron biofortified millets have the potential to provide additional bioavailable iron into the diets of young children and women of reproductive age, respectively. However, when implementing such approaches, acceptance in the target population is crucial and the potential negative impact of malaria infections on long-term iron absorption needs further investigations.

2.3 Zinc absorption from zinc (bio) fortified sorghum, millet and rice diets
Fortification of cereal staples with zinc is recommended to combat zinc deficiency. To optimize zinc absorption, strategies are needed to overcome the inhibitory effect of phytic acid (PA) and perhaps polyphenols (PP). A series of zinc absorption studies in adults investigated the effect of the following factors on zinc absorption from maize and sorghum porridges fortified with zinc (as ZnSO4 or ZnO) to levels of ~3 mg/meal: the addition of the phytic acid-degrading enzyme phytase immediately prior to consumption of the test meal; the ability of EDTA at different EDTA:Zn molar ratios to enhance zinc absorption from water soluble (ZnSO4) and water insoluble (ZnO) zinc compounds; the influence of sorghum polyphenols on zinc absorption in the presence and absence of phytic acid; and the ability of EDTA to overcome any potential inhibition of zinc absorption by polyphenols. The results showed that adding phytase to the maize porridge immediately prior to consumption increased the absorption of zinc as ZnSO4 by 82% (from 8.7% to 15.8%; P < 0.001); adding Na2EDTA at an EDTA:Zn molar ratio of 1:1 increased the absorption of zinc as ZnSO4 by 32% (from 10.0% to 13.2%; P = 0.01) but had no influence at higher EDTA:Zn molar ratios or on absorption of zinc as ZnO; zinc absorption was slightly higher from ZnSO4 (10.0%) than from ZnO (7.3%); sorghum polyphenols had no effect on the absorption of zinc (as ZnSO4) from fortified, dephytinized sorghum porridges, but decreased zinc absorption by 21 % from phytic acid rich sorghum porridges (from 10.7%; to 8.4%; P < 0.02); and the combined inhibitory effect of polyphenols and phytic acid in sorghum was overcome by EDTA. In conclusion, ZnSO4 was better absorbed than ZnO; phytase used to degrade PA during digestion or during food preparation substantially increased zinc absorption from zinc-fortified cereals, EDTA at a 1:1 molar ratio modestly enhanced zinc absorption from ZnSO4 fortified cereals but not ZnO fortified cereals; and sorghum PP inhibited zinc absorption in the presence, but not absence, of PA.

Fortifying cereal staples with zinc is a potential way of increasing zinc intake in young children in developing countries. However, phytic acid (PA) naturally present in the cereals strongly decreases zinc absorption. A stable isotope zinc absorption study was carried out in young children to investigate the ability of the PA degrading enzyme phytase to improve zinc absorption, when added to a cereal meal immediately before consumption. Fractional absorption of zinc (FAZ) was estimated in 35 young healthy Burkinabe children using the double isotopic tracer ratio method with 67Zn as oral tracer and 70Zn as intravenous tracer, in a crossover design. The test meals were: (a) a millet based porridge containing 1.3 mg total zinc (native plus 1mg added as ZnSO4) with a PA:Zn molar ratio of 7.7; (b) the same porridge with the enzyme phytase added immediately before consumption. The exchangeable zinc pool (EZP) was determined in 20 of the 35 children as a potential indicator of individual zinc status. Adding phytase to the meal resulted in a 68 % increase in zinc absorption (from 9.5 ± 3.4% to 16.0 ± 5.1% (P < 0.0001)). The mean EZP was 3.6 ± 0.5 mg/kg. There was no correlation between the EZP and FAZ values for either of the two test meals. It was concluded that the addition of phytase at time of consumption can usefully improve zinc absorption from zinc fortified cereal based complementary foods.

Cereals can be biofortified with zinc genetically by plant breeding or biotechnology, or through soil fortification. Zinc absorption from cereals can be inhibited by high phytic acid (PA) levels. It is possible that native zinc in cereals, biofortification zinc, and inorganic zinc fortification compounds are influenced differently by PA and other dietary components and absorbed to different extents. A study was conducted to compare zinc absorption from zinc biofortified rice with that from zinc fortified rice. The biofortified rice was hydroponically enriched with zinc and was a surrogate for rice biofortified through soil fertilization. Zinc absorption from hydroponically grown rice, intrinsically labelled with 70Zn, was compared to rice fortified with 70ZnSO4. Both rice meals had a PA:Zn molar ratio of 12 and a total zinc content of 1.1 mg. Fractional absorption of zinc (FAZ) was measured using the double stable isotopic tracer ratio method in 16 young healthy adults who consumed a single meal of biofortified or fortified rice porridge at 4 weeks intervals in a crossover design. The mean FAZ from the biofortified rice was 25.1 ± 8.7%, compared to 20.8 ± 7.1% FAZ (P = 0.076) from the fortified rice. These results suggest that the zinc accumulated in the biofortified rice was readily released from the rice matrix and that its absorption was influenced by PA and other food components in a similar way to fortification zinc; and that rice biofortified with zinc by soil fertilization is likely to be as good as post-harvest zinc fortification as an intervention strategy to combat zinc deficiency.

The results from the zinc absorption studies provide several ways for optimizing zinc absorption from zinc fortified foods. The most important finding is that the phytase enzyme added to cereal porridges immediately prior to consumption can degrade phytic acid during digestion in both adults and children and can substantially increase zinc absorption. Its potential addition to micronutrient powders and lipid based supplements for home fortification may be of important public heath significance. Another potential additive for increasing the absorption of zinc fortification compounds is EDTA, although this only appears to improve absorption from soluble zinc fortification compounds such as zinc sulphate and not from insoluble compounds such as zinc oxide. EDTA was shown to overcome the combined inhibitory effects of phytic acid and polyphenols in zinc sulphate fortified sorghum porridges. Sorghum polyphenols alone however did not inhibit zinc absorption, but they intensified the inhibitory effect of phytic acid.
Concerning the choice of zinc fortification compounds, the results confirm that there is little difference in absorption between water soluble and water insoluble zinc fortification compounds. Adequate absorption can be obtained with water insoluble ZnO making cost and sensory properties the most important criteria for selection.
In relation to the absorption of biofortification zinc, the results relate only to the agronomic practice of biofortification by soil fertilization. Zinc absorption from intrinsically labelled biofortified rice grown in hydroponic culture was similar that from fortification zinc added to control rice in the form of zinc sulphate. This suggests that absorption of the biofortification zinc was similarly influenced by phytic acid and other food components as absorption of the fortification zinc. Biofortifcation of rice via soil fertilization with zinc would thus appear to be as good an intervention strategy to combat zinc deficiency as the conventional zinc fortification of foods.

3 Development of improved (traditional) processing methods of millet, sorghum and maize enhancing iron, zinc and vitamin A bioavailability and/or reducing inhibitory effects of anti-nutrient factors for improvement of micronutrient status of young children and women

3.1 Changes in micro- and macronutrient composition of pearl millet and white sorghum during in field versus laboratory decortication.
Traditional decortication of pearl millet and white sorghum by hand pounding or using a mechanical device were performed in Burkina Faso, and compared to abrasive decortication in the laboratory using the same kernel lots. Using some nutrients as histological markers, the decortication characteristics and nutritional composition (iron, zinc, phytates, lipids, Acid Detergent Fibre (ADF) fibres and starch) of decorticated grains were measured. Decortication had numerous effects on grain composition but no significant differences were observed between the two traditional methods of decortication. The effects varied according to the type of grain mainly due to the fact that more germ was removed in sorghum than in millet, as the millet germ is more embedded in the endosperm. During abrasive decortication, zinc and lipid losses increased rapidly due to removal of the germ, particularly in sorghum. Phytates were shown to be located mainly in the bran and germ but also in the endosperm in millet. In both sorghum and millet, half the iron was removed when only 10% of grain dry matter (DM) was abraded. The method of decortication, shock or friction vs. abrasion, influenced the fractions removed and thus the chemical composition of the decorticated kernels.

3.2 Potential of non-GMO biofortified pearl millet (Pennisetum glaucum) for increasing iron and zinc content and their estimated bioavailability during abrasive decortication
Two non-GMO biofortified and one traditional pearl millet varieties were compared in abrasive decortication studies to evaluate their potential for increasing iron and zinc content. The phytate-to-mineral ratios were used to estimate mineral bioavailability. Iron and zinc contents in the biofortified varieties Tabi and GB8735 were two to threefold higher than in the traditional variety. Iron content reached 7.2 and 6.7 mg per 100 g DM in the biofortified varieties, which corresponds to the target values of biofortification programs. Zinc content was, respectively, 5.6 and 4.1 mg per 100 g DM in the GB8735 and Tabi varieties. Because of the presence of phytate and other chelating factors that were only partially removed during decortication, there was no improvement in iron bioavailability in the biofortified varieties. But whatever extraction rate, phytate-to-zinc ratios ranged between 6 and 18; zinc absorption could be improved by using these biofortified varieties for food processing.

3.3 Changes in iron, zinc and chelating agents during traditional African processing of maize: Effect of iron contamination on bioaccessibility
The effect of the different unit operations of processing traditionally used to produce four maize foods commonly consumed in Africa on the nutritional composition of the products was investigated, using Benin as a study context. The impact of the processes on lipid, fibre, phytate, iron and zinc contents varied with the process. The lowest IP6/Fe and IP6/Zn molar ratios, the indices used to assess Fe and Zn bioavailability were obtained in mawè, a fermented dough. Analysis of maize products highlighted a significant increase in iron content after milling, as a result of contamination by the equipment used. Evaluation of iron bioaccessibility by in vitro enzymatic digestion followed by dialysis revealed that the iron contamination, followed by lactic acid fermentation, led to a considerable increase in bioaccessible iron content. Extrinsic iron supplied to food products by the milling equipment could play a role in iron intake in developing countries.

3.4 Iron Contamination during in-field milling of millet and sorghum
Nutritionally, contaminant iron in foods may lead to overestimation of the satisfaction of iron requirement while iron deficiencies remain a widespread health problem. Iron contamination was measured in millet and sorghum grains after decortication and in-field milling using different equipment in Burkina Faso. Total iron content did not change significantly after decortication, probably due to a balance between losses resulting from the removal of iron-rich peripheral parts and contamination. Total iron contents increased significantly after mechanical milling irrespective of whether iron or corundum grindstones were used. Contamination was highly variable, ranging from 3 to 6 mg iron/100 g DM, and was mainly due to wear of the milling equipment. After in vitro digestion of traditional cereal dishes prepared with iron-contaminated or uncontaminated flours, the contaminant iron was found mainly in the insoluble fraction. Only in sorghum was a small proportion (4%) bioaccessible, showing that contaminant iron has poor nutritional interest.

3.5 Contribution of plant-based sauces to the vitamin A intake of young children in Benin
A food consumption survey on 420 children was conducted in four areas in Benin to identify the local vitamin A (VA)-rich foods most frequently eaten and assess their contribution to the coverage of VA requirements of young children. Mangoes, eggs, red palm oil, various leafy vegetable (LV) sauces and palm nut juice sauce appeared to be the main VA-rich foods consumed. The recipes of the most promising sauces were characterised. Sauces with red palm oil/palm nut juice showed high carotenoid contents ranging from 0.9 ± 0.3 to 4.0 ± 0.8 mg Retinol Activity Equivalent (RAE) /100 g dry matter (DM). Lipid contents were also high (from 39.6 to 66.8 g/100 g DM). When consumed, and taking into account the mean quantity eaten per meal, LV sauces containing red palm oil or palm nut juice contributed to the meeting of more than 70% of the recommended VA intake of young children.

3.6 Effect of a multi-step preparation of amaranth and palm nut sauces on their carotenoid content and retinol activity equivalent values
The preparation of a traditional sauce based on amaranth leaves cooked palm nuts or red palm oil (RPO) in Benin was described. The recipes included an optional step of leaf blanching at 100 °C, heating the RPO or boiling the palm nuts and finally cooking all the ingredients together. The influence of this multi-step preparation on the carotenoid content of the final dish was measured. During blanching of amaranth leaves, violaxanthin was the only carotenoid to be significantly affected by the thermal treatment. Retinol activity equivalent (RAE) remained high after blanching even when alkaline traditional potash was added. Heating the RPO was the most critical step because it considerably and very rapidly (in <3 min) decreased alpha-carotene, beta-carotene and RAE values (more than 70%). Sauces calling for palm nuts, RPO and amaranth leaves were equally advantageous in terms of final RAE value. These ingredients and sauces can thus be used in programmes to reduce vitamin A deficiency.

3.7 Fate of phytochemicals during malting and fermentation of Type III tannin sorghum and impact on product biofunctionality
The aim of the study was to assess the effects of sorghum bioprocessing into Gowee on iron bioavailability and antioxidant properties of the final products. Gowee is an African sour beverage, whose process combines malting and fermenting of sorghum grains. The effects of the durations of germination and fermentation on the phytochemicals were evaluated using a central composite design. The antioxidant capacity and iron bioavailability of the derived flour were also evaluated. During the germination process, the tannin content of the grain decreased from 429.5 to 174.1 mg/100 g DM, while the total phenolic content increased from 300.3 to 371.5 mg GAE/100 g DM. The phenolic acid contents of the flour were significantly modified as a result of the durations of germination and fermentation. Both germination and fermentation enhanced the antioxidant capacity of sorghum flour, and antioxidant characteristics were significantly correlated with the levels of total phenolics, tannins, and phenolic acids. Phytate content of sorghum grain decreased drastically from 1003 to 369.1 mg/100 g DM when the duration of germination or fermentation increased. This was associated with an increase in the bioavailability of iron.

3.8 Effects of processing on the nutritional value of sorghum based complementary porridges
This study evaluated the effects of process operations on the nutritional values of three types of sorghum porridges. These are fermented sorghum porridge, a composite porridge I (sorghum, soy and fish) and a composite porridge II (sorghum, bean and peanut). From nutritional point of view, the fermented sorghum porridge contains: proteins: 6.7%, fat: 2.3%, and total minerals: 1.17%; the composite porridge I contains: proteins: 21%, fat: 5.4%, and total minerals: 3,14%; the composite porridge II contains: proteins: 17%, fat: 0.98%, and total minerals: 2,6%. Soaking of sorghum grain for 23 hours, sieving after grinding and fermentation lead to a significant decrease in total phenolics and anthocyanins contents of the derived porridge.

3.9 Potential of leafy vegetables for increasing iron intakes and status
A stable iron isotope study assessing the iron absorption from traditional and improved leafy vegetable (LV) sauces was carried out in Switzerland, with 18 young women recruited among the students or staff in Zurich University. Burkinabe composite test meals consisting of maize paste tô either accompanied by the iron-improved amaranth leaf sauce, the iron-improved jute leaf sauce or the traditional amaranth leaf sauce were eaten as multiple meals for 2 days. The three LV sauces were prepared using the same batch of ingredients in the technological hall of DTA, in Ouagadougou (Burkina Faso) and kept frozen until their utilization in Zurich, in Switzerland. The maize was also purchased on a Burkinabe market, decorticated, soaked, washed and sundried before transport to Montpellier where it was milled.
Caution was taken to avoid any iron contamination during processing. Maize tô was prepared every day in the Zurich lab during the study. Iron absorption was measured as erythrocyte incorporation of stable iron isotopes. Fractional iron absorption from test meals with iron-improved LV sauces did not significantly differ (4.9%; P = 0.9324) resulting in a comparable quantity of total iron absorbed (679 µg vs. 578 µg; P = 0.33). Fractional iron absorption from meals with traditional amaranth sauce (7.4%) was higher than from the meals with the two iron-improved sauces (P = 0.0178 and P = 0.0145 respectively). However, the quantity of total iron absorbed from the meals with traditional amaranth sauce was not higher (591 µg; P = 0.3982 and P = 0.7308 respectively. Results indicate that LV sauces significantly contribute to iron needs; however, further measures such as adding iron absorption enhancers to the sauces, decreasing the concentration of inhibiting phenolic compounds in the sauces should be investigated to increase iron absorption.

4 Safety and efficacy of iron-fortified maize to alleviate anaemia in malaria-endemic areas in sub-Saharan Africa

The new flour fortification legislation in Kenya now ensures that pregnant women receive iron through a combination of fortified foods and universal iron supplementation. In view of the findings of the Pemba trial (Sazawal S, Black RE, Ramsan M, et al. Effects of routine prophylactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: community-based, randomised, placebo-controlled trial. Lancet 2006;367:133-43) indicating that supplementation with iron and folic acid to prevent anaemia increased morbidity and mortality, INSTAPA aimed to assess whether the iron intake resulting from this policy is safe. It hypothesized that flour fortification alone would reduce the risk of Plasmodium infection in pregnant women as compared to combined supplementation and flour fortification. The general objective of the study was to assess the effects of combined fortification of whole meal maize flour with iron (NaFeEDTA) and iron supplementation on safety indicators, and on iron status, in pregnant women.

4.1 Antenatal iron supplementation, Plasmodium infection and birth outcomes in Kenyan women: a randomized trial
Whereas coverage of antenatal iron supplementation is low and benefits are uncertain, there is evidence that it can increase the burden of malaria, with potentially devastating effects on maternal and neonatal health outcomes. We aimed to measure the effect of iron supplementation during pregnancy on maternal Plasmodium infection assessed at delivery, birth weight, gestational duration, fetal growth and maternal and infant iron status. In this study, 470 rural Kenyan women with singleton pregnancies, gestational age 13 - 23 weeks and hemoglobin concentration >= 90 g/l were randomized to supervised daily supplementation with iron (60 mg as ferrous fumarate) or placebo until 1 month postpartum. To prevent severe anemia, both groups additionally received 5.7mg iron/day through flour fortification. Intermittent preventive treatment (IPT) against malaria was given as usual. Plasmodium infection was assessed at birth by dipstick tests, PCR and histological examination of placental biopsies. We found no effect on Plasmodium infection risk (both intervention groups: 45%; difference, 95%CI: 0%, - 9% to 9%). Iron supplementation increased birth weight by 143 g (95%CI: 58 - 228 g) and reduced the prevalence of low birth weight (< 2,500g) by 65% (95%CI: 13% - 86%). The effect on birth weight was restricted to a pre-specified group of women who were initially iron-deficient (250 g; p-interaction = 0.008) and seemed achieved mostly through better fetal growth. Iron supplementation resulted in improved maternal iron status at 1 month postpartum, and enhanced neonatal iron stores. In conclusion, coverage of universal antenatal iron supplementation under cover of IPT must be increased. Antenatal iron supplementation leads to large improvements of birth weight, fetal growth and infant iron stores, with potentially immense benefits for infant survival and health that should outweigh any possible concerns about risks of malaria. Scaling up universal iron supplementation, even in the presence of universal iron fortification of staple-foods, in pregnancy in developing countries is likely to generate major public health gains.

4.2 Antenatal iron supplementation and serum concentrations of non-transferrin bound iron in pregnant women in a malaria-endemic region of Kenya: a randomised controlled trial
Supplementation with ferrous iron salts can lead to an increased incidence of malaria and other infectious diseases in children, which is possibly mediated by non-transferrin bound iron (NTBI). This study aimed to measure the influence of ingestion of a supplement with iron on serum NTBI concentrations in pregnant Kenyan women. Additionally, we assessed the influence of iron status, gravidity, maternal age, Plasmodium infection, HIV infection, alpha+-thalassemia genotype on the serum NTBI response to iron. Kenyan women with singleton pregnancies, gestational age 13 - 23 weeks and hemoglobin concentration was <90 g/l randomly received a supplement with iron (60 mg, as ferrous fumarate) or placebo. Blood was collected at baseline and at 3 hours after ingesting the supplement. Women were allowed to eat lunch between ingesting the supplement and second blood collection. Serum NTBI concentration was measured at using a flow cytometry-based assay. At 3 hours after ingesting the supplement, NTBI concentrations were similar between groups (mean for both: 0.18 μmol/l; difference, 95%CI: 0.01 μmol/l, –0.03 μmol/l to 0.05 μmol/l). Adjustment for possible confounders did not substantially change the effect estimate. We found no evidence that the NTBI response to iron was also not influenced by Plasmodium infection, iron deficiency, iron deficiency anaemia or alpha+-thalassaemia. In conclusion, this study does not support changing existing policies for daily supplementation with 60 mg iron as ferrous fumarate, at least in women at a gestational age between 13 and 23 weeks. Further studies are needed to determine the effect of iron supplementation on NTBI in pregnant women in a fasting state in various stages of pregnancy, especially the third trimester of pregnancy when transferrin receptor concentrations are highest. Research into gold standard methods to assess NTBI is urgently needed to enable accurate assessment of NTBI.

4.3 Diagnostic utility of zinc protoporphyrin to detect iron deficiency in Kenyan pregnant women
Zinc protoporphyrin (ZPP) has been used as a screening marker to manage iron deficiency in children and pregnant women and to estimate prevalence of iron deficiency. This study examined associations between ZPP and disorders that are common in Africa (Plasmodium infection, HIV infection, alpha+-thalassemia). We also assessed the diagnostic utility of ZPP, either alone or in combination with hemoglobin concentration, in detecting iron deficiency defined as plasma ferritin <15 μg/l. Analyses were based on a survey among Kenyan women with singleton pregnancies, gestational age 13 - 23 weeks and hemoglobin concentration >= 90 g/l. Linear regression analysis was used to identify factors that were associated with whole blood and erythrocyte ZPP. In women without inflammation, Plasmodium infection or HIV infection, we used Receiver Operating Characteristics (ROC) curves to assess the ability of ZPP to discriminate between women with and without iron deficiency. Lastly, we assessed the diagnostic performance of ZPP as a dichotomized variable, using conventional cut of points and thresholds calibrated to produce unbiased estimates of the prevalence of iron deficiency. Whole blood ZPP and erythrocyte ZPP were mostly determined by iron markers, and little influenced by inflammation, Plasmodium infection, HIV infection and alpha+-thalassemia genotype. ZPP had limited ability to discriminate between women with and without iron deficiency (ROC analyses, highest area-under-the-curve: erythrocyte ZPP, 0.73). Combining each of these markers with hemoglobin concentration had no additional diagnostic value. Conventional cutpoints for whole blood ZPP can result in gross estimates of the prevalence of iron deficiency, particularly when the true prevalence is low. ZPP has limited diagnostic utility, whether used a single diagnostic test or when combined with hemoglobin concentration

5 The effect of maize-based complementary foods fortified with iron and zinc on cognitive and psychomotor development of young children

The overall aim of this study in INSTAPA was to examine the potential of a nutritional intervention which is safe enough to be given daily to infants from 6-18 months of age living in poverty in a malarial area to enhance their social, emotional and cognitive development.
Using a randomized, controlled, double-blind trial, and the project enrolled infants less than 6 months of age from those identified in selected villages in the catchment area of a primary health care centre in coastal Kenya. At 6 months of age, the children were randomized to two treatment groups, an intervention and a control group. The nutrition intervention was one sachet per day for one year containing 5g of micronutrient powder (MixMe micronutrient powder, DSM, Switzerland) based on the WHO/FAO requirements, 2004. This powder has been approved for use in Kenya to fortify complementary foods. For iron, the formulation concurred with the conclusions of the WHO expert consultation in Lyon in 2006. These conclusions state that in malaria-endemic areas where malaria control programmes, are not optimally implemented and individual screening for iron deficiency is not possible, additional iron can be provided to older infants and young children as processed complementary foods. The control arm received sachets containing 5 g of powder containing the micronutrients but without the iron. These powders were added to finely-ground maize meal in the household for infant feeding; the maize meal was provided to all participating families by the project. The mothers of the participating children were shown how to prepare the food at home (see Table 1).

To avoid spillage of the product to other children in the household, each household received 2 kg of maize meal per week in the first 6 months of supplementation, and 3 kg per week for the second 6 months. Breast-feeding was promoted at all times. Distribution points in the vicinity of the health centre served as collection points for the food supplement, and the monitoring of health and motor milestones.

The intervention lasted one year so from 6-18 months of age. A battery of measurements were used for child assessment (growth, activity levels, and motor-, social-, emotional- and cognitive development), complemented with health measurements and maternal and home measurements. In collaboration with clinic staff, the children were followed up for a period of 24 months. The growth and development of the children from 6 months of age was used to determine the influences upon outcome at 12, 18, 24, 30 months of age.

5.1 The Micronutrient and maize meal supplementation was beneficial to the haemoglobin levels
There were no significant differences in haemoglobin levels between the groups at 18 months of age with respect to haemoglobin. However, when the INSTAPA children were compared to an not-supplemented group from the same area there was a significant differences between the two samples (p < 0.001) at 12 and 18 months of age during the time that the INSTAPA families were receiving micronutrient sachets and maize meal weekly. By 24 months of age the difference between the two study samples had narrowed, and they remained similar with respect to haemoglobin at 30 months of age.

5.2 The additional iron improved the health status and behaviour of the children during the supplementation period
Appetite: Over three quarters of the INSTAPA children were reported to have had a good appetite throughout the study, and there is no evidence that good appetite was linked to the child’s intervention group.
Growth: Stunting (height for age) increased over the study period from 6 to 30 months of age, as did the incidence of low weight for age. By contrast, mean head circumference for both the supplementation groups was above the international averages (WHO) for the supplementation period (6 to 18 months of age).
Sleep patterns: Children’s total sleep patterns particularly during the day were influenced by anaemia at 10 months (p = 0.027) 12 months (p = 0.029) and at 18 months of age (p = 0.048). Children’s total sleep patterns were influenced by anaemia at baseline, with anaemic children having fewer hours of sleep and more disturbed sleep (p = 0.03).
Motor Milestones (World Health Organisation Motor Milestone Chart): The children who received any iron during the supplementation period appeared to be at a disadvantage in achieving the motor milestones: the children in the non-iron supplementation group achieved the milestones at a younger age from Milestone 7 (Crawl) onwards, with Milestones 10 (Walking Supported) showing a trend at p = 0.06 and, Milestones 11 and 12, (Standing Unsupported and Walking unsupported) at p = 0.02 and p = 0.058 respectively.
Activity Levels (Naturalistic Observation): The children receiving the iron supplements were less active than the children from the non-iron group during the intervention at 12 (p = 0.043) and 18 months of age (p = 0.011) but there were no significant group differences 24 and 30 months of age.
Social communication (Vocalisation): There was a significant difference between the iron and control groups at 12 months of age, with the control group vocalising more, but the difference between the groups narrowed thereafter.

5.3 Differences between groups after the end of the supplementation period
The Kilifi Developmental Inventory (KDI) consists of two types of assessment, the Locomotor Score and the Eye-Hand Co-ordination Score, each consisting of several items, and together the scores are totalled as the Total Psychomotor Score. At baseline the influence of anaemia in each of the scores was examined, with no statistically significant differences found between the children with anaemia and those without. There were also no significant differences in six-monthly assessments between the experimental and control groups throughout the intervention period and up to 24 months. However, at 30 months there was significant difference in the eye-hand co-ordination score (p = 0.049) and the total psychomotor score (p = 0.040): the children who had received the iron fortified micronutrient for any period during the intervention scored higher than the control groups.

5.4 Understanding variability in child development outcome in resource-constricted settings: the contribution of the home environment
The main purpose of this study was to establish the manner in which proximal processes and distal contexts individually and collectively influence child development, and the way these relationships change at different ages. The effects of socioeconomic status (SES) and the home environment were explored through two different studies: sub-study-1 among a rural school-age population (the school-age study) and sub-study-2 among a rural infant population (the infant study). Structural equation modelling was used as the analysis method to examine the relationships among the variables of interest. Based on the bio-ecological theory, it was hypothesized that proximal processes within the home environment would have a stronger impact on child outcomes than the distal factors within the children’s contexts. The findings provided partial support for the theory. For the two groups, different causal pathways of background variables were illustrated through structural equation modelling. This study has made important contributions to illustrating which aspects within the home environment have the strongest impact on child outcomes. We recommend based on the findings of the current study that these aspects should be considered when planning interventions to improve outcomes for children living in resource-constricted settings.
The key findings of the current study are summarised to emphasise the contribution made to the current body of research. For one, the current study has demonstrated the reliability and validity of tools that have been modified for this study context. Their applicability to the study of children’s outcomes suggests that they can meaningfully be used to show how children are functioning in various domains.
The magnitude of the influence of background factors on outcome seemed to vary across ages. Some of the variables were more influential at younger ages, while others became important at older ages. These findings support the fundamentals of the theoretical model applied through this study that there are factors which are experienced directly by the child and are therefore more influential, while others are found within the child’s context, and may be less important. This information is useful in making decisions on which variables to consider at the various ages when looking at the influences on child outcomes.
Even though the data did not fully support the theoretical model, it was demonstrated how the theory could be meaningfully applied within the study context. While some of the aspects of child functioning may be strongly influenced by cultural factors, the findings demonstrate the universality of some of the aspects of child functioning.
Through the comparison of findings from infant and school-age populations, the study provided evidence of the interactions between SES and the home environment at younger and older ages. At both ages, there is strong evidence to support the association between proximal processes and distal contexts of the child. Even within relatively homogeneous SES settings, differences were observed in the home environments of children. Such information will enable the identification of families that are most at risk, based on children’s developmental outcomes.
Potential Impact:
The potential impact of INSTAPA is mainly related to the strategic objectives of INSTAPA as mentioned in the Annex I of the Grant Agreement.

Significant contribution to the improvement of the dietary quality of very young children living in resource poor areas of developing countries resulting in long-term health effects and a major step towards the Millennium Development Goals set for 2015

In a region characterized by consumption of monotonous staple food based diets leading to insufficient intakes of essential micronutrients iron, zinc and vitamin A among young children and women, INSTAPA research results do offer options for improvement of especially the staple foods cassava, millet, sorghum and maize. In formulating food-based dietary guidelines or agricultural strategies, the following findings could lead to an improvement of the nutrient quality of the food produced and consumed:
- Pro-vitamin A biofortified or yellow cassava is available with sufficient beta-carotene levels that are generally well accepted. Production and consumption of yellow cassava instead of white cassava would offer an alternative option for reducing vitamin A deficiency in sub-Saharan Africa.
- Iron biofortification of pearl millet is a promising approach as consumption would double the amount of iron absorbed of iron absorbed.
- Biofortification via soil fertilization with zinc appears to be as good and intervention strategy to combat zinc deficiency as the conventional zinc fortification of foods.
- Genetic diversity of iron and zinc concentrations of sorghum exists but at present the potential contribution of sorghum varieties to iron and zinc intake is not sufficient to solve iron and zinc deficiency.
- The use of NaFeEDTA seems to be the most feasible and promising approach increasing iron bioavailability from iron-fortified sorghum foods containing both phytic acid and inhibitory polyphenols.
- In remote areas with limited access to conventionally fortified foods, in-home fortification with millet-based complementary foods using enhancers, such as phytase and ascorbic acid, is a promising approach to provide additional iron.
- Concerning zinc fortification of cereal staples, zinc sulphate is slightly better absorbed fortificant than zinc oxide, while EDTA is a modest enhancer of zinc absorption. But adequate absorption can be obtained with zinc oxide making cost and sensory properties the most important criteria for selection.
- Phytase enzyme added before consumption can significantly increase Zn bioavailability (same extent as dephytinization) in adults and children. Its potential addition to micronutrient powders or lipid-based nutrient supplements for home fortification may be of important public health significance.
- Due to the low mineral contents and high contents of factors inhibiting absorption in sorghum and millet and to the observed effects of decortication (co-elimination of desired and undesired compounds), the optimisation of the contents in bioavailable iron and zinc in millet or sorghum-based dishes through improvement of processing is probably not sufficient. Leafy vegetable sauces significantly contribute to iron needs; however, further measures such as adding iron absorption enhancers to the sauces, decreasing the concentration of inhibiting phenolic compounds in the sauces should be investigated to increase iron absorption.

Concerning the safety of the studied food-based approaches and the functional consequences of especially iron fortification and supplementation, INSTAPA provides important results for public health.
- Asymptomatic malaria parasitemia decreases dietary iron absorption through low-grade inflammation. This may contribute to iron deficiency or iron deficiency anaemia or may blunt efficacy for fortification programmes in malaria-endemic areas.
- Antenatal iron supplementation leads to large improvements of birth weight, fatal growth and infant iron stores, with potentially immense benefits for infant survival and health that should outweigh any possible concerns about risks of malaria. Scaling up universal iron supplementation, even in the presence of universal iron fortification of staple-foods, in pregnancy in developing countries is likely to generate major public health gains.
- Addition of micronutrient powders to maize flour is beneficial to hemoglobine levels of young children and improved health status and behaviour, often only detectable at long-term interval.

Establishment of an international leading role of the consortium in the fields of biofortification, fortification and processing to increase the supply of bioavailable micronutrients from African staple foods by bringing together scientific and technological excellence

The Consortium comprised scientists from European University and Research Institutes from the Netherlands, France, Switzerland and United Kingdom as well as from African Universities and Research Institutes from Mali, Burkina Faso, Benin, Kenya, Nigeria and South Africa, and HarvestPlus. The Consortium was very strong and partners collaborated well in several work packages.
Through the collaboration in the Consortium, other mutual collaborations started and enabled partners to get access to follow-up research funding. For example, within workpackage 6 a successful collaboration of UNSA and ETH was established in an NIH programme. Also further collaboration of ETH and UAC, of WU, HarvestPlus and IITA, etc was established.
Partners of the Consortium were often requested to represent INSTAPA in several national and international meetings such as the Global Donor Platform on Rural Development, the CGIAR Science Forum on Nutrition and Health Outcomes: targets for agricultural research), EU High Level Meeting on meeting on European Investments in Biofortification; Nigeria Sorghum Transformation Value Chains Workshop, Food Fortification Panel Workshop of the African Nutrition Society, US NIH Technical Working Group on Iron and Malaria, EASTWESTFOODS, the IFPRI2020 Conference leveraging Agriculture for Improving Nutrition and Health.
Furthermore, often the Consortium was asked to be represented during official visits op diplomats of Embassies.

Partners of the Consortium published many scientific papers in double refereed scientific papers and presented both oral and poster presentations at important international Conferences such as the International Conference on Nutrition of the IUNS in 2013, the ANEC in 2011, 2012 and 2013. The scientific papers are often cited in other scientific papers and reports.

Capacity building in knowledge and skills to solve micronutrient deficiencies in sub-Saharan Africa and Europe

Capacity building in knowledge and skills has taken place at several levels. In total, 8 PhD’s finalised their research and training within the INSTAPA project; 3 from Europe and 5 from Africa. Four PhD dissertations were completed and successfully defended:
- W Amoussa Hounkpatin December 2011: 'Assessment of the potential of sauces accompanying staple foods in Benin to meet Vitamin A requirements of young children'.
- H. Fatoumata-Ba May 2012: 'Retention and bioavailability of iron and zinc during processing of traditional dishes prepared from local and biofortified cereals and consumed by young children in Burkina Faso'.
- C. Cercamondi May 2013: ‘Improving iron nutrition from sorghum and millet based Diets in malaria endemic areas’;
- M Brnic September 2013: ‘Optimizing zinc absorption from zinc fortified cereal staples’.
Three PhD dissertations are submitted or ready to be submitted and defence is expected before July 2014:
- Martin N Mwangi: ‘Safety and efficacy of iron interventions in African pregnant women’;
- Elise F. Talsma. ‘Yellow cassava: Efficacy of provitamin A rich cassava on improvement of vitamin A status in Kenyan schoolchildren';
- Kadzo-Wekulo: 'Understanding variability in child development outcome in resource-constricted settings: the contribution of the home environment'.
One PhD thesis is ready in draft form but need further refining before submission and defence:
- Jane Njenga. ‘Long-term Effects of a Low-Iron Multiple Micronutrient on Children’s Anaemia, Morbidity, Nutritional Status, Growth Trajectory, and Motor Development (South Coast – Kenya)’.

During the project period, 70 MSc, BSc and Internship students from Africa and Europe were also trained in INSTAPA linked to the different studies in the work packages.

Based on a standardised work plan, training needs assessments were carried out in Benin, Burkina Faso and Kenya among SMEs, local NGOs and local food processors. Workshops with the stakeholders were organised to discuss and finalise the results of the training needs assessment. Based on the assessments a two-week training module was designed entitled: ‘Towards solutions of micronutrient malnutrition’. A Teacher and a Student handbook were developed for professionals in SMEs and local food processors. Based on a short technical workshop in Benin discussing comments and remarks of a review committee, the training modules were finalised and an English and French version were developed. In 2011 the French version was of the training was implemented in Benin among 20 trainees from Benin, Mali and Burkina Faso. Based on the experiences the training module was slightly adapted and another field testing was done in Kenya in 2012 with 22 trainees from different regions in Kenya. Based on the second field testing, the training module was finalised, and printed in Burkina Faso. The final training module was launched at an INSTAPA workshop in Ouagadougou in November 2013. The training module is now used in training modules offered in the framework of FINSA (Formation Internationale en Nutrition et Sciences Alimentaires) of the Faculté des Sciences Agronomiques, Université d’Abomey-Calavi. Also other organisations use (part) of the modules for their courses or staff trainings, as well as University of Nairobi is using (part) of the modules to improve the curriculum.

Communication within and beyond the consortium regarding the ethical and scientific issues of concern to the agricultural and health sectors as well as consumers, enabling African populations to define and choose diets for optimal health for their children

Internally, communication within the Consortium was supported by the internal website (www.instapa.org). Throughout the project period, the internal website was continuously updated through the ‘Training and Symposia’, the ‘Research News’ as well as the ‘Press releases”. The announcements have been published and respective brochures and links made available. The home-page was half way improved with ‘Latest News’, ‘reminders’ and ‘Publication list’, ‘Thesis list’, as well as ‘Presentations and posters list’. In total 69 members registered to the internal website.

Through internal technical workshops and meetings, partners within a work package informed each other about activities and this enabled them to align activities but also to being informed about related activities outside INSTAPA. The 4 Newsletters published at the internal and external website also played the role of exchanging information between partners and outside the Consortium. The annual meetings taking place every year organised by alternating partners within the INSTAPA Consortium (in Switzerland, Benin, Kenya, South Africa, the Netherlands) enabled all partners to be informed about the progress of the different work packages. At several annual meetings, other experts were invited of related projects to mutually inform about work carried out.

In order to disseminate and present the major findings and knowledge of the INSTAPA project and its work packages to the broader (scientific) public, it was planned to organise an International Conference in one of the partner countries. At the same time, it became clear that in October 2012 the Nutrition Congress Africa (NCA) would be organised in Bloemfontein, South Africa. The theme of this Congress was “Transforming the nutrition landscape in Africa”. As this Congress aimed to reach a comparable audience as the INSTAPA Conference but had a much larger budget to reach out to the scientific community, it was decided to organise a parallel session within this Nutrition Congress Africa to present the INSTAPA results. This enabled INSTAPA to disseminate the results in the region where the studies were carried out and where the results should be used and implemented, reaching a wide range of scientists, professionals, decision makers and practitioners from the region. The dissemination is planned to be complemented with a more regional oriented seminar on INSTAPA in West-Africa (Burkina Faso) and with an international oriented dissemination during the International Congress of Nutrition in Granada in 2013. The NCA 2012 was a joint venture a joint scientific meeting of the 24th congress of the Nutrition Society of South Africa (NSSA), 12th congress of the Association for Dietetics in South Africa (ADSA), and the 5th African Nutrition Epidemiology Congress (ANEC V) of Africa Nutrition Society (ANS) and was held from October 1-4, 2012 at the University of the Free State under the leadership of Dr Ronnette Lategan and her team in the local organising committee. The parallel INSTAPA session was organised within the theme “Community Level: Health and Disease”. The aim of the parallel meeting was to present the latest results of the work packages 1-6 and to disseminate the brochure as well as the recipe booklet and the training module in printed form. INSTAPA representatives from the work packages 1-6 presented their latest findings in dedicated presentations. After the presentations a lively discussion took place on INSTAPA and its results. During this session approximately 75 persons attended. The complete presentations are available in D7.10s. The contribution of the many INSTAPA scientists to the NCA in the form of presentations, posters but also during the discussions in the different sessions was much appreciated.
In addition to this international workshop, two local workshops (in Kenya and Burkina Faso) were organised. A dissemination workshop took place in November 2013 in Kenya, Kibwezi, during which the results of all studies with biofortified cassava in Kenya were communicated to study participants, parents, schools, community leaders and local government officials. Furthermore, a 2-hour seminar was held at the Kenyan Agricultural Research Institute (KARI), during which the scientific results of the CASSAVITA study, which was carried out under the INSTAPA project, were presented. Further, the way forward to release yellow cassava varieties in Kenya was discussed, which KARI will now take up. It is estimated that it will take some further breeding studies over a period of 3-5 years to accomplish this. A national workshop was organised in Burkina Faso by IRSAT-DTA in November 2013 to present main results and achievements obtained in Burkina Faso through work package 4. This workshop was attended by 46 participants.

Results of the INSTAPA project were further disseminated in the form of posters, abstracts for scientific conferences, and oral presentations during scientific conferences. In total, 23 oral presentations have been delivered and 25 posters. Key presentations were given at the NCA Congress in Bloemfontein (October 2012, see above), at the International Conference on Nutrition in Granada (September 2012), the International Congress on Hidden Hunger in Stuttgart (March 2013), and the CGIAR Science Forum 2013, Bonn (September 2013).
Also, although more towards the end of the project, 10 scientific papers have been published in peer-reviewed scientific journals and more than five are submitted awaiting reviews, while there are many still in draft form to be submitted in 2014. Also a book chapter was written on neuropsychology of children in Africa. A book on improving the mineral contents in nutrition of young children’s diets in the West African Sudan Savannah through biofortified varieties of sorghum is ready in draft form and will be published soon.

A booklet in English and French with recipes and descriptions of commonly prepared and consumed sorghum and millet based dishes and accompanying sauces is published. This booklet identifies and characterise the processing methods and nutritional value of the main traditional foods consumed in Benin and Burkina Faso. The information in the booklet forms a good basis for nutrition interventions in the West African region and provides information on dietary intake and food patterns to policy makers. The booklet is distributed through the partners and during several conferences and international meetings. The booklet is in high demand and also often heralded for providing information on diets and dishes that is often lacking for this region.
In order to render the results of the INSTAPA project useful and especially to reach the people who do not have access to the modern tools of communication such as internet, `Space for Change approach’ (approach based on participatory and interactive modes of communication) was used to design or develop a methodological framework of innovative communication tools for promoting the innovations and results developed in INSTAPA. These include video development, popular journal articles, and popular posters. Within this framework, the following was developed:
- EuroNews documentary of INSTAPA. In September 2012 EuroNews broadcasted the documentary on INSTAPA as a coproduction between EuroNews, INSTAPA and the European Commission. The documentary was 8.5 minutes long and the broadcasting started just before the World Food Day, 16th of October 2012 and was repeated 22 times thereafter. It was broadcasted in 130 countries in Euro New eleven broadcasting languages (English, German, Arabic, French, Italian, Russian, Spanish, Turkish, Portuguese, Ukrainian and Persian; link accessed 18th of November 2013. The link to this INSTAPA documentary is accessible from the external and internal INSTAPA website.
- A second video documentary was made specifically for WP6 focused on field impressions on cognitive development in Kenya (http://www.instapa.org/everyone/2198/5/0/20 ). A link to the video is available at the internal and external INSTAPA website.
- A third video video documentary was developed by Martijn van Beenen specifically focusing on the PIMAL study within WP5. This documentary also included a detailed description of the local fortification and this part can be used to illustrate the developed business model (http://vimeo.com/86523239 ). Additional video material was delivered that can be used for educational purposes. A link to this documentary is available at the internal and external INSTAPA website.
- A poster was developed for specifically WP2 to disseminate the results of the efficacy trial of yellow cassava to the schoolchildren, their parents and the teachers involved. These posters were lefty behind at the schools so that the children, parents and teachers could still refer to it whenever useful.
- A popular article and a brochure on INSTAPA was written and published in ‘Project Magazine' (available at the external and internal website of INSTAPA) in October 2013: .
- Attention on the Harvest Plus website on results of INTAPA research:
http://www.harvestplus.org/content/study-finds-strong-preference-biofortified-cassava-eastern-kenya &http://www.harvestplus.org/content/study-finds-iron-rich-pearl-millet-can-meet-full-iron-needs-children.

Monitoring Ethical approvals
As the INSTAPA project comprised interventions studies in human subjects carried out in Kenya, Benin and Burkina Faso which include blood withdrawal, children 0-12 years, the Grant Agreement stated that the protocol needed to be approved by required Medical Ethical Committees at local and national level of each research side, and by an acknowledged Medical Ethical Committee affiliated with one of the European partners. All approvals from Ethical Review Committees in Benin, Mali, Kenya, Burkina Faso, South Africa, The Netherlands, Switzerland, and United Kingdom were received (see Table 2). Acquired approvals were immediately upon receipt by the coordinator communicated to EU and uploaded on the INSTAPA website.

Exploiting new scientific knowledge in order to strengthen the competitiveness of local SME (farmers, millers, and food processors) as well as African and European food industry targeted at evidence-based production of healthier complementary foods for African children

The new flour fortification legislation now ensures that young children and women receive iron through fortified foods. However, most people in rural area depend on own grown grains, mill these at the local miller and as such have no access to fortified flour. As a first step to study the potential of an SME at local level, a business model was developed for local iron fortification of maize flour in Kenya. The business model was developed based on the experiences with local fortification at posho-mill level as introduced during the study of work package 5. The business model developed takes as an example the establishment of a commercial venture that will aim at iron fortification of whole maize flour at village mill level in the rural areas of Kisumu district. The venture will be blending and packing premixes purchased from the GAIN Premix Facility with locally produced (industrial) maize flour into sachets with pre-blend that can be used for fortification at village mill level. Each sachet will contain 20 g pre-blend that can be used to fortify 1 kg of flour. The sachets will be distributed to village millers who will fortify their own sales of maize flour and/or fortify the maize flour of their customers as a service. The venture will further ensure quality assurance, provide training and marketing services to millers and support education to end customers. The business has a ’social enterprise’ character and therefore the ownership (individual or group) should adhere to ’social entrepreneurship’ principles. To start the business initial investment support from a donor or NGO programme would be required to the amount of approximately €50,000, excluding Technical Assistance in training and start up. In the first years of developing the business, it should be closely linked to an NGO programme aimed at improving household food security and nutrition that would assist in popularizing the concept of local fortification. Such a linkage would ensure that the local community will remain involved and safeguard the continuity of the business. During the development of the business model, discussions have been held with representatives of the Micronutrient Initiative (Kenya), the Food Fortification Project of the Ministry of Public Health and Sanitation, Division of Nutrition, and with representatives of the small and medium food Industries like GAIN Kenya, Unga Holding Ltd, Fortitech, and the Consumer Information Network. Feasibility of the business model was judged based on the pilot carried out within the work package 5 and these experiences have been included in the business model description.
Friesland Campina, the largest dairy industry in The Netherlands, has expressed an interest in reviewing our results, especially related to WP5, with a view to possible patenting. In response, LSHTM and WU have entered an agreement to jointly file a patent, and intend to have a meeting shortly with this industry (and perhaps other industries) to review the possibility of licensing industry to use this patent insofar as this does not impairs access of poor people in developing countries to iron interventions. Under the agreement between LSHTM and WU, both the costs of patent filing and possible revenues will be shared on a 70%:30% ratio. Because of the patenting issue, results have been shared so far with colleagues on the basis of a confidentiality agreement.
List of Websites:
www.instapa.org
Dr. Inge D. Brouwer
Division of Human Nutrition
WAGENINGEN UNIVERSITEIT
P.O. Box 8129
6700 EV Wageningen
T: (+)31 317 485920
inge.brouwer@wur.nl