Food and Agriculture COST Action FA0905: Mineral-improved crop production for healthy food and feed

Transcription

1 Food and Agriculture COST Action FA0905: Mineral-improved crop production for healthy food and feed Workshop: Improving the composition of plant foods for better mineral nutrition Session 1 Chair: Emmanuel Frossard, Rapporteur: Aghili Forough In the first secession the most important critical nutrient elements in European population and the strategy to improve food quality and also to increase the nutrient absorption by human was discussed. The summary of the presentations that was given in this session are available below: 1) Alexa Meyer, University of Viena, Astria The first presentation was given by Alexa Meyer, University of Viena, Astria and she spoke about Mineral needs in Europe as compared to the global situation. Nutrient intake in Europeans is high in fat, sodium and cholesterol and low in fibers, Ca, I, Vitamin D and Fe (mostly in women), that is related to low consumption of fruit and vegetable due to more availability and also lower price of energy-dense but nutrient-poor foods than the nutrient rich food. It is often recommended to reduce red meat intake. However, red meat is a good source of iron, especially soluble haem that is best available. Iron and Zinc deficiency is more frequent in overweight especially in children. The nutrient status in human is dependent on age and also gender. For example elder people have lower Zn intake due to lower food consumption. Pregnant woman have low intake of Fe, I and K during their pregnancy. These countries also have a severe Se problem (86.5% of participants had Se plasma levels < 125 µg/l, the threshold for maximal GSH Px activity) because the availability of Se is low in soil. One strategy for fighting with nutrient deficiency is food fortification. However, mandatory fortification is not widely practised in Europe but, voluntary fortification of some foods is more or less common, especially in breakfast cereals. As a conclusion: Fe, I, Se and zn are critical nutrient elements in Europe. Intake does often not correlate with status. Fortification and diverse diet can help to improve nutrient status in human. 2) Richard Harrell, ETH Zurich, Switzerland The second presentation was given by Richard Harrell, ETH Zurich, Switzerland and he spoke about Composition of plant foods and mineral nutrition.

2 Plant-based diets are one important reason for trace element deficiencies in poor people. Major intervention strategies to correct micronutrient deficiencies are food fortification, supplementation, diet modification and biofortification. Breeding, engineering or agronomic techniques are biofortification strategies that aim to produce staple crops that are dense in micronutrients. Plant composition has a major influence on iron and zinc absorption by humans. For example Phytic acid, Ca and phenolic acid for Fe are the major inhibitory factors and ascorbic acid, organic acids muscle proteins, inulin and carotenoids are major enhancer factors. As a conclusion phenolic compounds, together with phytate, are the major inhibitors of iron absorption in our diet. Ascorbic acid and other enhancer factors increase Fe absorption and consumption of food with high amount of these components and low phenolic component and phytic acid is very useful to increase the absorption of Fe. 3) Ann-Sofie Sandberg, Chalmers university of Technology, Gotenborg, Sweden The third presentation was given by Ann-Sofie Sandberg, Chalmers university of Technology, Gotenborg, Sweden and she spoke about the influence of phytic acid on mineral nutrition. Between 1-3 % in whole grain cereals, nuts and legumes is phytic acid. Phytic acid is inhibitor of some nutrients such as Ca, Mg and Fe. Foods component are eefctive on nutrient absorption in positive ( promoting component) and negative (inhibitors) ways. Phytic acid is an important inhibitor for Ca, mg, Fe and Zn. Ascorbic acid and protein meat incraese Fe absorption and animal protein is known as promoter for Zn absorption. Inosytol-pantaposphate is an inhibitor for Fe and Zn absorption and Inosytol-Triposphate and Inosytol-tetraposphate have inhibitory effects on Zn absorption in processed food. One strategy to decrease the concentration of phytic acid is breeding aprouch to produce the garin with least amoun t of phytate. Another aprouch could be degradation of phytate in food with adding phytase to the food. Redusction of phytate in diets witch contain milk is not sufficient to improve fe and zn absorption due to the reducing of Fe absorption by Ca and also formation of insoluble component. As a conclusion Dephytinization is useful when the food doesn t contain polyphenols or if it isn t consumed with milk. inhibiting effect of phytate is counteracted by intake of ascorbic acid (Fe) and meat protein (Fe, Zn) in the same meal. 4) Ines Egli, ETH Zurich, Switzerland

3 The last presentation of this session was given by Ines Egli, ETH Zurich, Switzerland, who gave an inside into the influence of polyphenols on mineral nutrition. Polyphenols is heterogeneous group of molecules derived from the secondary plant metabolism and can be found in beverages, fruits, vegetables, cereals and legumes in concentrations, which can vary strongly. For human nutrition polyphenols can have positive effects (e.g. antioxidant, preventing effects on cancer, etc) as well as negative effects by reducing the bioavailability of proteins and minerals. Polyphenols build very stable complexes e.g. iron in the food and intestinal tract, which significantly decreases the absorption. Common beans, which are an important staple food in many countries, can have a high content of iron but also a high content of iron absorption inhibitors as polyphenols and phytic acid. These antinutrients are mostly situated in the seed coat. The interaction effect between polyphenols and phytic acid has shown that both components have to be reduced to enhance the absorption of iron. Sorghum polyphenols also have a strong inhibition effect on the iron absorption. The impact of polyphenols of sorghum on the iron absorption can be partly overcome with the addition of NaFeEDTA and fully with vitamin C. Concerning zinc, it seems that the phytic acid content of the sorghum is determining for the absorption. In the presence of phytic acid, the absorption can also be enhanced with the addition of EDTA. Session 2 ( ) Ines Egli, chair of the second session, welcomed everybody to the afternoon session, briefly talked about its organization and introduced the first speaker. Maria Andersson, ETH Zurich, Switzerland Iodine nutrition in Europe and iodine bioavailability from plant foods Maria Andersson first presented that goiter was a problem for many years in Europe and that in 1993 still 110 countries were affected by goiter, the main reason being insufficient iodine in soil and thus in the diet. She also mentioned that goiter was a real public health problem in Switzerland.

4 She then explained that iodine is an essential component of the thyroid hormones and that low intake of iodine results in multiple adverse effects, collectively known as iodine deficiency disorders (IDD). The daily iodine requirements as well as the level of excess intake were presented followed by the consequences in the thyroid in case of insufficient intake which leads to thyroid dysfunction. She emphasized that the thyroid hormones are the most critical hormones during pregnancy as they are essential for brain development of the fetus. She showed that the native content of iodine in food sources is low and that the major sources are bread (containing iodized salt), milk and dairy products (due to dietary supplements to cows, or use of disinfectants prior to the milking of the cows). Seaweed and sea foods contain a lot of iodine but are not widely consumed in Europe. Vegetables and fruits have a low content. A widely used strategy to prevent iodine deficiency is universal salt iodization. Salt is fortified with mg iodine/kg salt by adding KI or KIO 3 during salt production. Salt is consumed by everybody daily in constant amounts. Packaging is important to prevent losses of iodine. Recent data show that in 128 countries households are using iodized salt, but coverage is sometimes very low. However, today, 71% of people are consuming iodized salt, which increased by 20% since Iodine status has to be monitored closely in order to provide enough iodine and to prevent excess intake. The used biomarker is urinary iodine concentration measured in spot urine samples which reflects immediate iodine intake, is sensitive, is a population (and not an individual) indicator due to high day to day variation and therefore requires a large sample size. Global estimates of iodine status derived from large national or sub-national, cross-sectional studies in school children are available from 150 countries (97%) and showed that 32 countries are still iodine deficient and that in 11 countries iodine intake is to high due to excessive iodine in salt. The prevalence of deficiency was estimated to be 30%, with half of these children living in iodine deficient countries and the rest in pockets of sufficient countries. The prevalence of sever deficiency is only 5%. In Europe, 11 countries are still mildly deficient with a prevalence of 30% which is mainly due to the increased consumption of processed foods for which the food industry does not use iodized salt. In order to prevent coronary heart diseases salt consumption should be reduced which however is not a problem for salt iodization as levels can be adapted. Alternative interventions to iodization of salt are iodine in bread, cooking oil or mineral water. Iodine in the food is either protein bound or present as I - or IO 3 - and is taken up in the intestine. However the mechanism is not clear. Some active uptake may take place but mostly seems to be passively diffused. It is difficult to assess bioavailability and two balance studies with a controlled diet have shown that an increased intake leads to an increased excretion and suggests a bioavailability of 92 and 95%. Some nutrients interactions including goitrogens (inhibiting iodine absorption), iron, vitamin A and selenium deficiency (due to the function in enzymes

5 which regulate the thyroid hormones) and environment pollutants (incorporation into thyroid) have been shown. Conclusion: mild IDD is still a public health problem. The native food content is low, but bioavailability is high and due to the close interaction with other micronutrients a balanced diet is important. Discussion: Food industry is reluctant as they think iodine deficiency is a problem from the past, they are not aware of the problem. Some countries have restriction for the use of iodized salt (Denmark in bread) Norway: milk very low in iodine, European laws allow supplements in cow feeds, but this is not enough Long term public health consequences: cognitive function due to development of brain in infants and neonatal phase, longitudinal studies underway (supplementation during pregnancy) Infants likely to be more iodine deficient than children or adults: infants are concerned as they should not consume salt before the age of 1 year. Breast milk concentration may not provide enough iodine because iodine status of lactating mothers may not be sufficient. Complementary food should be fortified with iodine. There is very little data. But study in Switzerland showed low breast milk levels and low iodine status in infants. Pierdomenico Perata Plant biofortification with iodine Iodine concentration is usually very low in plants with the exception of a few plants with very high concentrations (algae). In Japan, where the consumption of algae is common, IDD is not a problem. Some iodine fortified crops are available in supermarkets such as potatoes, tomatoes and carrots. Biofortification of spinach resulted in a high iodine content of 300 ug/100g but also in a reduction in biomass due to phytotoxicity. Although through biofortification of rice (by adding iodine to the water) increased plant iodine content by 2 fold, the amount in the seed did not reach levels to meet human requirements. Studies investigating the transport within the plant showed that xilemic transport is higher than phloematic transport and that root treatment resulted in higher iodine content than leaf treatment. Generally, physiology of iodine in plants has been little studied and knowledge with respect to uptake, transport and retention is limited. By

6 metabolic engineering the sodium iodine symporter (NIS) was integrated into Arabidopsis and these plants showed a higher uptake and a better distribution of iodine in the plant. Thus, this transporter is functional in plants. However, when higher amounts of iodine are fed to the plant iodine is not more accumulated which may be due to volatilization caused by the enzyme methyltransferase. This has been proven by plants in which the gene for this enzyme was knocked out (hol-1 mutant) and which showed better growth, higher iodine content and better iodine distribution. However, the hol-1 gene seems to be induced by the increased iodine uptake in NIS transgenic Arabidopsis, thus protecting the plant from too high iodine accumulation. But the cross between the NIS and hol-1 plants resulted in higher iodine levels. Conclusion: Volatilization plays a major role in reducing iodine content in plants. Thus selecting crops with low hol-1 activities (such as tomato) are favorable for iodine biofortification. Discussion: As of today it is not known how iodine is stored in the plant, but soon this will be known. Iodine in the form of iodide is taken up better but it is also more phytotoxic Timing of applying iodine in biofortified crops is crucial as it is phytotoxic Genomes for important crops (potato, rice, tomato) are about to be sequenced Algae is different from other plants, but a specific transporter is not known Erick Boy Working Group A: Targeted levels of minerals in plant foods / Biofortification versus post-harvest fortification Erick Boy started with defining the two terms of biofortification and post-harvest fortification and described the differences as well as the considerations of the two strategies. The most important issue with most strategies is that they should not affect organoleptic properties. In a next step he presented the program implementation steps for post-harvest fortification including the definition of the target population, the assessment of mineral intake and status, the selection of the fortificant as well as the food vehicle and the determination of the fortification level, followed by establishment of the regulatory parameters, costs, monitoring and finally marketing strategy. Fortification is not always applicable, and is difficult if food processing is decentralized and in regions with subsistence farming.

7 In order to establish the nutrient target level two methods can be used: the EAR cut-point method or the probability method. Both methods give a similar answer, and thus it is easier to use the cut-off method with the goal that 97,5% of individuals have an adequate intake without causing a risk of excessive intakes. Often interventions overlap which increases the risk of giving too much of a distinct micronutrient. The challenges that HarvestPlus is dealing with in order to develop and evaluate biofortified foods have been outlined with the estimation of the targeted micronutrient content as the central point during the development phase. To set the target increment of a distinct micronutrient in a distinct plant genetic variation (maximum), non-biofortified average baseline and the target level to have an impact in human is considered and then the amount to be added to the non-biofortified average to reach the target level is defined. Thus, for some micronutrient-crop combinations the agronomic biofortification was abandoned (e.g. iron in wheat, maize and rice for which the maximal genetic levels are below the target level). For such combinations the transgenic approach is the only option. The importance of testing the seeds in areas where they are planned to be grown was emphasized as yield could be substantially affected. About 10 years are needed until a biofortified crop is expected to be released. The breeding targets for iron in beans and millet and zinc in rice were illustrated and their contribution to the EAR in a specific population as well as the potential effect on prevalence if 35 or 70% of this population consumed the biofortified crop was shown. Claire Mouquet Working Group B: Influence of food processing on composition of plant foods Claire Mouquet started with the statement that for all micronutrients the amount absorbed is much smaller than the intake and that this bioavailability factor varies a lot ranging from 2 to 90%. The three main factors influencing absorption are the human body (nutritional and physiological status), the form of the micronutrient in the food and the food matrix. Basically, the origin of micronutrients in foods can be divided into intrinsic (natively present in the food) and extrinsic (contamination from soil, cooking equipment, fortification). In case of

8 biofortification we add some extra micronutrients by breeding, genetic modification or fertilizers. The intrinsic forms of a mineral are different. Iron exists as heme iron in meat and fish, as nonheme iron in the Fe 2+ and the Fe 3+ form which is found in animal source food and plant foods and we may have a third form which is ferritin-iron and for which we don t know how it is metabolized (absorbed intact or destroyed to non-heme iron and then joining the iron pool). There are many interactions of iron with the food matrix, which consist of inhibiting factors (phytates, polyphenols, proteins, calcium, oxalates) and activating factors (vitamin C, organic acids, muscle proteins). For others the effect is not clear (non digestible polysaccharides, vitamin A, carotenoids). Food processing has two types of effects: first on the mineral content as losses are expected during processing or increases due to contamination and second on the food matrix changes which influence the competition with other micronutrients, the content of chelating and activating factors, and oxidation and denaturation. The presentation was concluded with some examples of traditional food processing in Africa, such as the different effect of decortication (zinc losses depend on the shape of the grain as it is mainly found in the pericarp; iron losses are much higher than losses in dry matter as iron is concentrated in the peripheral part of the grain). The effect of traditional processing of millet on iron content was illustrated and the problem of contamination iron visualized. The contaminating iron is expected to be found in the raw millet, which means that true (intrinsic) iron content maybe best determined after washing and before decortication. Cooking resulted in a huge increase of the iron content due to contamination depending on the final product arising the question what role contamination iron plays in human nutrition (is it absorbed?) and if values in food composition tables are correct. Bioaccessibility studies in millet products have shown that only a small amount is dialyzable Fe and that contamination iron is mostly insoluble and that the small part that is soluble may not contribute too much. Conclusions: Processing effects on mineral content can simply be determined but changes due to effects on mineral bioaccessibility are complex and numerous, and it is difficult to develop a predictive model. Ann-Sofie Sandberg

9 Comparison of methods to assess mineral bioavailability (in vitro vs in vivo) In vitro methods are needed as they provide a fast screening tool. In a first step the content of micronutrients, inhibitors and enhancers are measured and in a next step bioavailability can be estimated using different methods. The simplest in vitro method is the dialysis technique which estimates bioavailability by measuring the solubility or dialyzability of minerals by mimicking the gastro intestinal conditions. With this method the correct direction of a response is given but not the correct magnitude as compared to human studies. A drawback of this method is that the reproducibility between labs is small as different conditions are used by different groups. In addition, large molecules (like heme iron) cannot be detected. The Caco 2 cell model is able to additionally assess the uptake into or through the enterocytes. After the two-step in vitro digestion the sample is transferred to the apical compartment and dialyzed through the membrane before the mineral can be taken up by the Caco 2 cells. Thus, iron uptake into the cells is used to predict bioavailability in humans. The cells contain all iron uptake and transport proteins but hepcidin is not involved. This system is useful for screening and molecular mechanisms of iron absorption can be studied. However, there is poor agreement between labs and the magnitude of response is not the same as in humans although the direction of response is correct. There are still many differences when this model is compared to human absorption studies. There is no ph gradient during digestion, no outer mucus layer, no blood to be transported and no control by hepcidin. In order to compare in vitro studies to human studies the foods should be prepared exactly the same way. There is only one direct comparison of maize and bean meals in which absorption from the maize meal was well predicted while absorption from the bean meal was not. Some animal models are useful, such as the suckling rat models to predict zinc absorption in humans. This model is less useful for iron as inhibitors and enhancers have a lower effect. Pig is a good model but cannot be used for screening. Human studies using radioisotopes or stable isotopes can sensitively measure absorption from a meal. The use of stable isotopes is safe but expensive and intrinsic labeling is a problem. Conclusion: In vitro studies can predict bioavailability in humans but results must always be confirmed in humans.

10 Francesca Sparvioli Iron fortification in bean: is the lpa1 mutant a good candidate? Francesca Sparvioli started with showing that iron deficiency is a major public health problem in young children and women also in developed countries and that strategies to reduce this deficiency are supplementation, fortification or biofortification either using transgenic or conventional breeding techniques. One possibility is to produce plants with a reduced phytate content. Phaseolus vulgaris L., the common bean, is the staple food for many people and is an important source of protein, is high in iron but also high in antinutrients such as phytates, lectins and raffinosaccharides. Phytate plays a role in plants and foods. In humans, it does not only have the negative effect of decreasing bioavailability of different minerals but it also has some positive effects (anticancer, antioxidant and anti-inflammation). As monogastric animals excrete phytate from the feed it accumulates in the soil. Thus, developing low phytate crops also reduces animal waste not only human malnutrition. In the common bean lpa1 mutant phytate content is decreased by 90% and raffinose by 25% and solubility trials of the iron have shown a 7-fold increase in free iron. The bean lpa1 mutant lines have low phytate/fe and phytate/zn molar ratios and ferritin formation in Caco 2 cells in mutants with white background was in increased but not with brown or black backgrounds. The advantages are the reduced phytic acid content in the seed and thus improved bioavailability of several micronutrients. However, in plants like maize, wheat, barley and rice these mutants show a reduced fitness up to lethality and a reduction of only 40-60% in phytate. Conclusion: lpa1 bean lines show acceptable agronomic performance and beans with white seed coats have significantly more bioavailable iron. But human trials are needed to confirm that the bean lpa1 is the first truly biofortified staple crop. Discussion: The beans will also be produced under local conditions Cross-breeding with high iron beans by CIAT is planned Promising results from human absorption studies

11 Lisa Miranda Influence of antioxidants from potato on iron bioavailability Lisa Miranda introduced the topic by showing how widespread iron deficiency still is. Potato is the 4 th most important crop worldwide and is rich in proteins, and antioxidants. Some varieties are also rich in Fe and vitamin C which makes it a crop to be considered for partially solving the iron deficiency problem. The most important polyphenols found in potato are chlorogenic acid and rutin. The effect of those on iron uptake into enterocytes was tested using an in vitro digestion model with Caco-2 cells and finally assessing ferritin content in the cells. Ferritin formation was increased when increasing concentrations of pure FeSO 4 was added to the cells which was further increased by adding ascorbic acid. Adding increasing concentrations of FeCl 3 had no impact on ferritin synthesis but with the addition of ascorbic acid an effect was seen. Thus, the Fe 3+ form first needs to be reduced to the Fe 2+ form. When chlorogenic acid was added to Fe 2+ or Fe 3+ with ascorbic acid ferritin synthesis decreased. Rutin addition increased ferritin synthesis dose dependently. Adding rutin and ascorbic acid in combination showed a synergic positive effect if not treated with too high levels of ascorbic acid. The digestion of two potato varieties (Vitelotte, rich in chlorogenic acid and Nicola, containing rutin) showed a decreased ferritin expression if a digested extract of Vitelotte was used probably due to the higher chlorogenic acid content. Conclusion: The effects of the polyphenols take place in the intestinal lumen. Chlorogenic acid chelates iron and antagonizes iron. Rutin probably reduces Fe 3+ to Fe 2+ which can then be taken up by DMT1. Discussion: No quercetin is formed Session 3 - Chair: Rainer Schulin, Rapporteur: Markus Lenz Oral presentations: 1) Influence of plant composition on selenium bioavailability, presenting author: Martin Broadley University of Nottingham, UK

12 M Broadley presented work on Se biofortification with emphasis on studies conducted in Malawi. First biofortification field experiments (conducted from ) made use of different Se formulations in liquid and granular form. Linear response of Se grain concentration vs. application rate was observed when selenium was given in form of selenate. M Broadley then continued presenting the case of Malawi, where human energy supply is mainly via maize (more than 50%) and animal products are hardly present in the diet (around 60 calories per day and person). Soil chemistry would suggest higher Se availability at elevated ph values. This is underlined by plasma Se levels in a pilot human study conducted in 2 Malawian villages living on low / high ph soils. M Broadley concluded with an estimation of Se biofortification costs if these were to be included in the Malawi fertilizer subsidy scheme introduced in ) Biogeochemistry of selenium and food chain quality, presenting author: Helinä Hartikainen, University of Helsinki, Finland H Hartikainen reported on the complex interplay that determines transfer of Se from bedrock to soil to plant to animal / human populations, i.e. the Se cycle. Although Se is not considered essential to plants, some may hyperaccumulate Se, such as Astragalus bisulcatus and Morinda reticulata. In Finland, first reports on adverse effects of Se deficiency are as old as from 1933, although these effects were not recognized as being due to Se at that time. H Hartikainen stated that milk Se content may be the most sensitive parameter to monitor the effect of Se fertilization, since the Se content of cereals may be influenced by imports. H Hartikainen suggests that also plants may benefit from Se, although it is considered not to be essential for plants. Some still unpublished results suggest that Se promoted the accumulation of starch and soluble sugar in plants and that it may retard plant senescence. Sequential extractions of soils that had received 12 years of Se fertilization showed that the major fraction of Se was associated with organic matter and some 30% of Se was bound to recalcitrant fractions. 3) Short communication: Bioaccessibility of selenium: Se-enriched leek (Allium ampeloprasum var. porrum) versus food supplements, presenting author: Gijs Du Laing, Ghent University, Belgium G d Laing presented results on bioaccessibility of Se enriched leek produced by fertilization with either selenite or selenate in comparison to 2 Se food supplements, i.e. Se Precise (Se yeast) and ACE-Se tablets (SeMet). The bioaccessibility was assessed after passage through a simulated gastro-intestinal system. A microbial consortium taken from so called Simulator of the Human Intestinal Microbial Ecosystem (SHIME) reactors was used to study the role of microbes in the last part of the colon. In comparison to food supplements, Se in leek showed higher bioaccessibility. Differences may have been due to the presence of fillers and binders in the food supplements.

13 4) Short communication Selenium biofortification on bread making wheat in Badajoz (Spain), presenting author: Maria Poblaciones, University of Extremadura, Spain M Poblaciones presented the results of a trial studying Se fertilization at different application rates and its influence on the Se content of wheat used for bread making in Spain. The soils of the two experimental sites (Cordoba, Badajoz) had total Se levels classified as deficient to marginal, and also the bioavailable Se fractions were inadequate to produce food/feed sufficient in SeM Poblaciones discussed how unusual weather conditions may influence the results of such field trials. Results of the working group discussions: - Biofortification is now limited to one nutrient per crop. What about multiple biofortification and the food basket approach? A balanced approach for biofortification is needed, there is not one size that fits all (i.e. industrialized vs. developing countries. Rice is low in most nutrients and may be particularly suited for multiple biofortification with nutrients such as iron, zinc, and vitamin A. Concentrate on plants that already have high contents and focus on having each particular food to contribute to the total food basket Bio-engineering is technically possible, but may face resistance / acceptance problems (there is resistance to fertilizing techniques as well)-> Educational component - Should improvement in bioavailability of minerals from biofortified crops and mixed diets be a priority? The combination of foods and ingredients is context-specific and the number of combinations can be very large and therefore direct research on each combination is not practical Mathematical models (algorithms) are available to estimate fractional and total mineral absorption that can provide sufficient guidance, if the necessary data on nutrient and antinutrient composition is available for the entire diet in question. Iron absorption prediction algorithms could be improved to increase their accuracy. Zinc algorithms require more data from studies with children. - Is it possible to accelerate biofortification?

14 One can accelerate biofortification by improving fertilization schemes. An additional screening of existing crops would be useful. Tools that can be used: Modelling is useful to provide a framework. Sensitivity analyses and economic modelling can help to design packages of interventions that are best suited in a given socioeconomic context (this area needs development). Strategy should be integrated: Focus on improving mineral levels (plant breeding, fertilization), mineral absorption (i.e. malting, etc) and increasing nutrient enhancers (promotion of fruit consumption) - Policy advocacy necessary to increase investment in biofortification research with a global perspective of food security, rather than a regional-focused funding strategy, particularly in Europe Decision has already been taken to allocate a large proportion of foreign assistance to global food security, responding to current demographic trends and the implications of population growth on food production. How to access these funds is not clear yet. - Which side effects result from biofortification? Agronomic performances need to be taken into account. Reducing pythate by reducing P fertilization will result in yield decrease; reduction of polyphenols may increase disease susceptibility. Effect on acceptability: decrease of phytate may affect final food quality (e.g. texture) Compromising efficacy of biofortification itself: Increase in Fe may require P- fertilization that will result in increased phytate content Increase of mineral content by conventional breeding or fertilization is probably limited, however, nutrient distribution and form of biofortified foods might differ from those of fortified food Session 4 ( ) Richard Hurrell, the chair of the fourth session, welcomed everybody to this last session and introduced the first speaker. Nicola Lowe

15 Systemic review of different factors influencing zinc bioavailability Nicola Lowe started with illustrating why it is important to improve zinc bioavailability from a global perspective. Severe stunting, wasting, and intrauterine growth restriction are responsible for 2.2 million deaths in poor countries. In Pakistan where stunting, underweight and zinc deficiency prevalence is high, many child deaths are linked to malnutrition. Main staple foods are rice and wheat and most people do not consume milk, only rarely meat but a lot of vegetables and pulses. Thus, total dietary zinc intake is enough but bioavailability is too low. In order to improve zinc bioavailability complementary food quality needs to be improved, more animal products should be consumed, but they are not affordable, supplementation would be an option but is not sustainable, fortification, biofortification and food processing are other options. Within the EURRECA (European Micronutrient Recommendations Aligned) project a systematic review of factors influencing zinc absorption was done and a part of the work was presented. The project was set up with the goal to harmonize the approach to setting recommendations for zinc, iron, iodine, folate and vitamin B12 intake. For zinc dietary reference values (DRVs) are very variable due to different expert groups and different methodologies. The focus was on zinc absorption, factors affecting zinc gains and losses, intake-status relationships and health outcomes. The factorial approach for setting DRV s is based on the basal losses, the requirements for maintenance and growth and the bioavailability factor. In the case of zinc, dietary factors can act at the level of absorption but also at the level of reabsorption of endogenous zinc. The search protocol for the systematic review included Ovid Embase, Ovid Medline and Cochrane Central and reference lists from reviews in order to pick up all relevant papers. Human studies were divided into the following categories: adults and elderly, pregnant and lactating women, children and adolescents and infants and data were extracted and meta-analysis done. For the group of elderly and adolescents the following dietary components were found to potentially influence zinc absorption: phytate, micronutrients, zinc, proteins, human milk vs. cows milk vs. whey based formula, oxalate, fat/cholesterol, tea, maillard browning and dairy. Twenty papers were selected in relation to the effect of phytate for the meta-analysis. Comparing fractional absorption of zinc form high versus low phytate diets showed that absorption is 11% higher from low phytate meals. Comparing high phytate:zn ratio meals (>15) to low meals (<15) showed a higher absorption in the low ratio meals. When grouping by single meal vs diet no difference was found.

16 Conclusion: Phytate is a potent inhibitor of zinc absorption with a 14% reduction in zinc absorption from a high phytate meal. The use of modeling techniques/algorithms may be useful for setting DRVs for zinc. More data are required for other potential inhibitors and strategies to increase the content and particularly the bioavailability of zinc are needed. More data for the infant population are important. Discussion: The impact single meal vs. diet is not different How to define Zn bioavailability? Different to absorption? Yes, bioavailability is absorption and utilization; absorption can be measured, bioavailability is difficult to measure. Measure retention by radioisotopes and stable isotopes. New DRVs for Europe by EFSA? EFSA will use the recommendations put together by EURRECA All studies have been taken into account for the meta-analysis irrespective of the method to assess absorption Philip White Biofortification of Edible Crops with Zinc According to the Copenhagen consensus 2012 the greatest concern lies in vitamin A, iodine, iron and zinc deficiencies. Many soils are alkaline and therefor have a low Fe, Zn, Cu and Mn availability. If the element is not in the soil crops can t have it and thus, it needs to be applied as fertilizer in order to improve the plant. Plants are like humans as they also have homeostatic mechanisms to control zinc levels. Zn travels from the leaves to the grain. Genetic variation is high in leafy crops but low in roots, seeds and tubers. Brassica oleracea was used as a model crop. Field and glasshouse trials with plants grown with low and high P and low and high Zn fertilizers have shown that with low P, Zn concentration in the edible tissue is higher and that Zn fertilization results in an increased Zn content. The genetic variation was found not to be very high but large differences were shown if grown in glasshouses or in the field. Important is that all genotypes do not behave in a similar manner under the same conditions (P and Zn addition). Phytotoxicity limits zinc biofortification but levels are high with 200 mg/kg for brassica and 500 mg/kg for spinach.

17 Potato is good tuber to be fortified with zinc as it is widely consumed in Scotland contributing with 5% to daily zinc intake. By increasing foliar Zn application more Zn ends up in the tuber but it is saturated. The same is seen in rice grains and is probably because of a protective effect of the phloem. A strong relationship between N and Zn concentration in the tuber was found. Zinc transport in the phloem seems to be the process limiting Zn biofortification of potato tubers. Zn is transported as Zn-nicotianamine and cereals overexpressing nicotianamine syntheses have higher Zn concentrations. Thus, Zn transporters are candidate genes to be targeted for transgenic plants. Combining agronomy and genetics lead to an increased Zn concentration in potatoes with some varieties reacting more than others. A lot of Zn was found in the skin of the control potato whereas in the biofortified one both, flesh and skin, contained higher Zn concentrations. Thus, such a potato would increase UK dietary Zn intake by 20% and would reduce the percentage of people below the recommended intake from 4% to 2.2%. Conclusion: Zn concentration in edible crops can be increased by agronomic or genetic strategies or a combination of both. Leaves have greater Zn concentration than seeds but leaf zinc concentration is limited by phytotoxicity, whereas seed and tuber concentrations are limited by phloem mobility. Minor QTL may be linked to genes for transport proteins. Discussion: Application of fertilizers simple as they can be combined with pesticides Surprisingly Zn oxide for foliar spraying went into the plant better than Zn sulfate, but commercial fertilizers were used which contain other ingredients Role of proteins: interaction sulfur nutrition and Zn? Not yet done, rather apply it as sulfate to detoxify Zn applied as foliar spraying: how much in plant and how much in soil? End of season, 30% of Zn is in or on the plant, thus 60-70% in soil; next crop in same plot, will residual be taken up? This is done currently. Shift in intake with biofortified potatoes: what impact to expect in young children. If infants benefit as well is not known. Costs for a biofortified potato program? Not done, only checked if it was possible and then do human trials; but additional costs for fertilizers are minor compared to the total costs of crop production; major fertilizer is 3% of crop production Correlation N and Zn in grain: could the increasing protein content have an impact on bioavailability? So far, only N is done but not the N containing compounds. May be an enhancer Zn bioavailability in green leafy vegetables: no study done.

18 Daniel Persson Zinc speciation in the cereal endosperm The central question of Daniel Persson s talk was, what new methods can bring for bioavailability and biofortification studies. His work focuses on method development for speciation analysis, which is on the borderline between element analysis and proteomics. The first step consists in a gentle extraction that keeps the zinc-binding intact. The second step is then a combination of size exclusion chromatography / anion exchange chromatography / ion pairing chromatography coupled with ICP-MS. The technic is multi-elemental. The major analytical challenges are the extraction in order to keep the metal binding intact. For the detection the challenge is that there are interferences. And the purity of the peak is important for the identification of Zn-binding proteins. The LC-ICP-MS methods have been applied to a range of plants (plant tissues/cell types/ligand types in rice and barley). With the extractability of Fe and Zn from the rice endosperm the main binding stages should be detected. Thus, first total Zn and Fe content is detected, than using phytase, amylase or protease bound Zn and Fe is released, thus this extractability is different. Zn was found to be mainly protein and starch bound whereas iron is primarily bound to phytic acid. Iron was co-eluting with phosphorus and zinc with sulfur containing proteins and copper. Why is native Zn binding important? For biofortification this may play an important role, as protein binding/composition might be a key factor to understand zinc bioavailability. Through biofortification zinc concentration increases but at the same time protein composition can change thereby influencing the bioavailability. Zinc sub-fractionation studies in rice endosperm showed that the water-soluble fraction was 17% and was well linked to albumins and globulins (20% of total protein in endosperm). 83% was water-insoluble and linked to protein body 1 which is mainly prolamins and protein body 2 which is mainly glutelins. Laser ablation-icp-ms can be used for localization but not speciation of zinc in order to check if zinc has reached the endosperm. Isolation of protein body 1 and 2 has been successfully performed. Conclusion: LC-ICP-MS can be used for soluble intact Zn-species analysis. Peak purity is important and often requires 2-3 dimensions of chromatography. The 4 main Zn-binding proteins

19 from the soluble fraction in the rice endosperm await proper identification. Over 80% of the Zn is found in the water-insoluble fraction, which consists mainly of starch and protein bodies. Discussion: More than 60% of total endosperm iron is bound to phytic acid, some was not extractable, no Zn found in this fraction How separate endosperm from seed: always some contamination of outer layer, but minor Extraction experiment: amylase had only an effect together with protease which leads to the assumption that proteins are on the surface of starch grains and that the major Zn fraction is in starch-protein complexes Zn-bound to proteins but not phytate, why negative effect in very refined grains? Zn is lost from the Zn-protein complex in the stomach, when the ph goes up Zn binds to phytate. The solubility of the Zn-protein complex may have an impact on Zn bioavailability. Paula Pongrac Zinc supplementation of wheat in metal polluted soil to increase nutrients and decrease pollutants in grains In North Slovenia soils are high in total Pb, Cd and Zn. Zn concentration of wheat grown in polluted soils are higher than average Zn concentration in wheat grown on normal soil. Wheat was fertilized with zinc in the nitrate form. Bulk concentration of elements in wheat grown on contaminated soil was assessed by MS techniques and spacial element distribution was measured by micro-proton induced X-ray emission (micro-pixe). In the whole grain Zn concentration increased from 90, which is higher than the average Zn content of grains grown on normal soil, to 220 mg/kg DW. Fe, Ca and Pb content increased also. Cd content decreased. When the grain is milled to flour this was the same but additionally K content increased in fertilized plants. Spatial element distribution images can show concentrations of P, S, K, Ca, Mn, Fe, Cu and Zn in fertilized and non-fertilized grains. Higher Zn concentration was found in fertilized crops in all peripheral layers. Fe and Zn are mainly found in the aleuron layer together with P and K. This is around 12 mg/kg in the endosperm for Fe and Zn and about 450 mg in the aleuron.

20 Conclusion: Addition of zinc fertilizer resulted in a higher concentrations of Zn (2.5-3 fold) in wheat grown on polluted soil. Cd and Pb concentrations were above the upper tolerable limit. The increase in Zn concentration was seen in different tissues. Discussion: Localization of Cd difficult to see with PIXE, as concentration is too low. General discussion In order to come back to the original aim of the workshop which was Improving the composition of plant foods for better mineral nutrition, Richard Hurrell asked the main speakers of the four micronutrients, iron, iodine, selenium and zinc to summarize the outcome. Iron (Ines Egli) The major problems with iron absorption are the inhibitors present in staple foods, particularly if diet consists predominantly of staples. Phytate has to be reduced to very low levels in order to have an impact on iron absorption (to 10 or less % of initial content), which is difficult to achieve with plant breeding. However, for beans this may be possible. Inhibition of iron absorption with respect to polyphenols depends on the crops. Most cereals do not contain relevant levels, but in beans and sorghum this is a problem. Ascorbic acid, the best iron absorption enhancer is not present in crops and susceptible to processing which doesn't make it feasible to be taken into account for biofortifying staple crops. Wheat is not a promising crop for Fe biofortification. Achievable levels are too low to make an impact on nutritional status of a population. Due to the localization of Fe in the grain using processing methods such as decortication and milling, not only the inhibitors but also Fe is lost. Thus, it is difficult to have a balance to keep most of the Fe in while reducing the inhibitors. Fermentation can help in that sense. With respect to speciation of Fe in crops, we do not know a lot. Fe is mainly bound to phytate but we don t know about other binders. Only a few crops are promising for biofortification with iron. Thus, biofortification should be combined with food processing.

21 Iodine (Pierdomenico Perata) Iodine biofortification of plants is a new topic, because there is almost no knowledge of the physiology of iodine in plants. Iodine has no physiological role in plants. However, iodine biofortification may be suitable to fill the gap of the people not reached by universal salt iodization. But it should never replace this strategy. There is room to integrate iodine biofortification into agronomic practices. Rice is not suitable for iodine fortification, but other crops do much better. We need better understanding of the physiology (uptake, transport in plant, storage). We don t know if storage iodine is bioavailable and we don t know how the food matrix affects bioavailability. Not only the agronomy but the selection of plants may be more important. Genetic modification may be the choice but as GMO is not welcome, exploiting natural variation is probably the solution. Within this new research area (iodine in plants), plant biologists and agronomists need to work together to provide a wide variety of iodine-fortified vegetables for developed countries in supermarkets. Selenium (Philip White) If there is a lack of selenium availability in soil, fertilizers are the solution as there is a good uptake of selenium with a linear relationship between dose of fertilizer and plant content. The easiest way is to use conventional fertilizers. Most of the Se is taken up by humans and it doesn t matter in what form it is in the plant. Bioaccessibility studies show that most forms are taken up very well. Attention should be taken with respect to species in biofortified crops as toxicity is reached quickly. Zinc (Philip White) Phytate is the main inhibitor of Zn absorption. Thus, in parallel of increasing Zn intake phytate decreasing strategies should be used. It is not known what the protein fraction does. But an increased absorption in presence of animal proteins has been shown. However, those are not easy to put into grain. It is important to keep studying the protein interaction. Zn is bound to cysteine and thus, increasing the cysteine content of proteins could be a promoter for Zn uptake. However, cysteine is genetically controlled, thus

22 this may only be possible by GMO. Targeted fertilization at the time when proteins are expressed may be possible. The identification of such time points is important. Only a small fraction of the fertilized Zn is taken up by the plants. Thus, we add more zinc to the soil through spraying, which will accumulate in the soil. The question is if this is sustainable and there is concern that we may contaminate the environment. Thus, the whole production system needs to be sustainable including factors such as water management, erosion control and not only looking at biofortification. But in that whole framework the aim is to increase micronutrient concentration in the plant. The solution would be to have plants with a genotype that take Zn up well in alkaline soils. Erick HarvestPlus is concentrating on increasing Fe and Zn in a few staple crops and in testing if sufficient levels can be reached with respect to human nutrition. Thus, HarvestPlus tests if the crops developed will result in increased nutritional status. Due to these results, target levels may be revised. In some crops inhibitors may be decreased. At the interim there is a consultation with Zn experts to revise Zn requirements used for setting the target levels of HarvestPlus. If target levels are increased HarvestPlus needs to talk to the plant breeders again to see if targets can be increased and to check the possibility if inhibitors can be decreased. One example is to cross the low phytate beans with the high iron beans. Work with ETH for further testing is in process. Multiple meal studies should be conducted. We need to establish the road map for breeders for the next 5 years. Evidence looks good but we need to bring in the reduction of inhibitors. Everybody needs to be convinced (plant scientists as nutritionists). Final words: Bal Ram Singh Some final words from Bal Ram Sing emphasized that the goal of this workshop was to focus on working groups 3 and 4, which was achieved by this workshop, and that we were able to have more interaction between plant and human nutrition scientists. During the workshop we heard very good presentations on biofortification and bioavailability/accessibility and human health and how this is interrelated. He thanked the organizers for a very successful meeting and mentioned that everybody is invited to participate in the next COST meeting in Lisbon. He then also thanked all speakers and poster presenters and finally all participants. He mentioned that the presentations will soon be available on the COST website.