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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Plant, Soil and Nutrition Research » Research » Research Project #433201

Research Project: Bioavailability of Iron, Zinc and Select Phytochemicals for Improved Health (Bridging Project)

Location: Plant, Soil and Nutrition Research

2019 Annual Report

1. In collaboration with international teams of plant breeders, we will develop lines of staple food crops such as beans, pearl millet, sorghum, rice, lentils and maize with improved Fe and Zn content and bioavailability. 1.A. Identify molecular markers and ultimately the genes that influence iron level and bioavailability in maize seed. Quantitative trait loci (QTL) mapping and an in- vitro digestion/Caco-2 cell culture model system will be used for this research sub-objective. 1.B. Identify molecular markers and ultimately the genes that influence iron level and bioavailability in lentils. Quantitative trait loci (QTL) mapping and an in-vitro digestion/Caco-2 cell culture model system will be used for this research sub-objective. 1.C. Identify agricultural and processing practices that enhance the nutritional quality of Fe in staple food crops. 1.D. Continue our work to identify compounds that promote or inhibit Fe bioavailability in staple food crops. 2. Evaluate a surgical duodenal loop model in poultry as a tool to measure bioavailability of health promoting phytochemicals. 2.A. Determine if absorption of selected phytochemicals can be measured in the poultry duodenal loop model. 2.B. Define bioavailability of specific phytochemicals from select foods using the validated duodenal loop model.

This project has two major objectives. The first is to develop lines of staple food crops with improved bioavailability and content of iron (Fe). To do so we will employ our established screening tools of an iron bioavailability bioassay and an animal model. In conjunction with these tools, we will make use of mass spectroscopy and marker assisted molecular breeding techniques. With these approaches, we will be able to identify and utilize regions of the plant genome that harbor genes associated with the enhanced food crop nutritional quality for Fe. To further complement the above approaches, we will continue our work on the identification of agricultural and food processing practices that enhance the bioavailability and content of Fe and Zn in staple food crops. The second major objective of this project is to develop a method to measure and evaluate the bioavailability of functional phytochemicals from plant foods. To do so, we will assess the use of a surgical model utilizing the unique intestinal anatomy of the broiler chicken to measure absorption of compounds from the duodenal segment of the intestine. This approach uses an established surgical preparation Involving anesthetized animals. Our initial efforts with this model will focus on compounds that are common to many staple food crops, fruits and vegetables. These phytochemicals will include the flavonoids epicatechin, quercitin, caffeic acid, chlorogenic acid and kaempferol. Enhancing our knowledge of bioavailability of health promoting phytochemicals will significantly improve efforts to develop more nutritious plant foods.

Progress Report
As of the writing of this report, our new project plan has been re-reviewed and only moderate modifications are needed. Researchers report on studies related to those objectives below. Improving the Yellow Bean Market Class for Fe Nutrition and Consumer Appeal: In collaboration with an ARS scientist in East Lansing, Michigan, the study conducts research on development of the yellow bean market class within the U.S. and abroad. Due to their fast cooking tendencies, yellow beans are often marketed at premium prices in the same geographical regions where dietary iron deficiency is a major health concern. Researchers also demonstrated that yellow beans are distinct from other market classes because they accumulate the antioxidant kaempferol 3-glucoside in their seed coats. In an animal feeding trial, researchers compared iron bioavailability of three faster cooking yellow beans with contrasting seed coat colors from Africa (Manteca, Amarillo, Njano) to slower cooking white and red kidney commercial varieties. Animals fed Manteca yellow bean diets had significantly higher Fe status than animals fed the white or red kidney bean diet. These observations capitulate a series of studies identifying the Manteca yellow bean as a candidate to address iron deficiency as it will be promoted by nutritional quality and fast cooking traits. United States consumers should value the fact that yellow beans can cook more quickly and can be a dietary source of kaempferol 3-glucoside, a functional food ingredient that is implicated for enhanced Fe uptake, and also linked to anti-cancer, antioxidant and anti-inflammatory properties. Investigating the Genotype, Environment, and Genotype by Environment Effects on Bean Fe Content and Bioavailability: A critical issue facing iron biofortification of beans is whether or not beans can be consistently bred to produce higher Fe content. In a unique study, researchers analyzed 16 bean genotypes containing yellow, white and red varieties that were grown across 9 locations, over 2 harvest years, spanning 3 agro-ecological zones in Uganda. These samples were evaluated for consistency of cooking time, iron concentration and iron bioavailability; thus, representing a definitive study to characterize the genotype (G), environment (E), and genotype by environment (GxE) effects on the above traits. Over two successive harvest years, cooking time was demonstrated to be a highly tractable trait across all varieties. Manteca yellow bean varieties consistently demonstrated the capability to deliver more absorbable Fe relative to red varieties that are equal or higher (approximately 20%) in Fe concentration. In contrast, Fe concentration values were highly variable, often differing 40-80% whereas Fe bioavailability was significantly more consistent. These results are significant as they indicate that breeding for high Fe concentration in beans is not a sustainable approach for bean Fe biofortification; however, breeding for high Fe bioavailability, appears to be a stable trait that can result in the delivery of more absorbable dietary Fe. Iron Bioavailability of Extruded and Fresh Bean Pastas: In collaboration with an ARS scientist at East Lansing, Michigan, the study evaluated the iron bioavailability of seven commercially available bean varieties with different cooking times and seed coat colors. The iron bioavailability of white and yellow bean flour ingredients formulated into fresh pasta was significantly higher than their whole boiled bean counterparts. This was due to the breaking of the cotyledon cell wall during processing into pasta flour, thus liberating more intracellular Fe for uptake. In addition, the iron bioavailability of fresh white and yellow bean pastas were superior to durum wheat pasta formulated in the same manner. The iron bioavailability of fresh pastas prepared with the darker colored cranberry (Etna), red (Red Hawk) and black (Zenith) beans was equal to or lower than the whole bean. The study attributes this effect to the polyphenols in the seed coat that are known to inhibit Fe uptake. In extruded pasta, high temperatures significantly enhanced the iron bioavailability of pasta from white and yellow bean varieties, while the iron bioavailability of pasta from red and black beans remained low. These observations are significant as bean flour products such as pasta are increasing in popularity both domestically and abroad as producers and consumers look for new pulse crop products with improved nutrition. Polyphenolic Profiles of Yellow Bean Seed Coats and Their Relationship to Iron Bioavailability: Polyphenolic compounds can promote or inhibit iron uptake. The study characterizes the relationship between iron bioavailability and polyphenol composition of seed coats in a panel of yellow beans selected to represent five Central and South American and Sub-Sahara African market classes. This panel also included two white and two red mottled bean lines, which represent high and low iron bioavailability in dry beans, respectively. Yellow bean seed types contained high concentrations of kaempferol 3-glucoside, a known promoter of iron uptake. The results are significant as correlation between the ratio of promoting to inhibiting polyphenols (P/I) and iron uptake was observed and could be used to predict the net effect of seed coat polyphenols on bean Fe bioavailability. Iron Fortification of Chickpeas: Chickpea was examined as a potential vehicle for Fe fortification. Iron fortificants were applied by a spraying and drying method onto split “desi” seed (dal), desi flour and kabuli flour. Significantly more bioavailable Fe was delivered in all of the fortified products with the desi chapatti samples having the highest levels of bioavailable Fe. Consumer acceptability tests demonstrated that NaFeEDTA-fortified cooked chickpea scored the highest among the fortificants. Although appearance of the fortified products changed with storage, no organoleptic changes occurred. This study demonstrated that chickpea products can be effectively fortified with acceptable consumer preference. Iron Bioavailability in Peas: In collaboration with Saskatchewan University, Canada, pea varieties with varied levels of phytate and carotenoids were tested in a feeding trial aimed to assess iron bioavailability. Pea varieties with lower phytate content and higher carotenoid content positively affected body weight, improved iron status, increased intestinal functionality and altered the intestinal microbiome composition and function. These results are significant as peas are popular foods for infants and toddlers, where Fe nutrition is critical. Identifying Genetic Regions of Wheat that Enhance Nutritional Quality of Iron: In collaboration with the National Institute of Agricultural Botany, United Kingdom, researchers screened mapping populations of wheat to identify genetic regions associated with iron bioavailability. A single genetic region was tentatively identified to significantly effect Fe bioavailability. If confirmed, this study gives wheat breeders an additional strategy to breed for improved Fe nutrition. Exploring the Role of Nicotianamine (NA) in Food Fe Bioavailability: NA is a compound in plants with high affinity for iron (Fe2+) and other divalent metal cations. In wheat, NA serves as the biosynthetic precursor to 2’ deoxymugineic acid (DMA), a root-secreted mugineic acid family phytosiderophore that chelates ferric iron (Fe3+) in the rhizosphere for subsequent uptake by the plant. Previous studies have flagged NA and/or DMA as enhancers of Fe bioavailability in cereal grain although the extent of this promotion has not been quantified. Thus, the study utilized the Caco-2 cell bioassay for Fe bioavailability to compare NA and DMA to two known enhancers of Fe bioavailability – epicatechin (Epi) and ascorbic acid (AsA). Both NA and DMA were found to be stronger enhancers of Fe bioavailability than Epi, and NA is a stronger enhancer of Fe bioavailability than AsA. NA reversed Fe uptake inhibition by myricetin (Myr; a potent Fe uptake inhibitor) more than Epi. These results are significant as they identify NA as a factor for improving Fe bioavailability in staple plant foods. Iron and Zinc Biofortified Wheat with Increased Nicotianamine (NA) Content: In collaboration with a wheat breeder from Melbourne University, Australia, assessment of iron (Fe) and zinc (Zn) bioavailability in a wheat variety with higher NA, Fe and Zn content vs. control wheat variety (ie. normal NA, Fe and Zn content) was conducted via a chicken feeding trial. The study demonstrated that NA holistically improved the chicken Fe status and positively altered the gut microbiome when delivered bound to Fe or as NA biofortified white wheat flour. Alterations to host intestinal functionality, hypertension related gene expression and feed energy conversion confirm NA’s role as a potent phytonutrient present in all plant-based foods. Effects of Soluble Plant Origin Prebiotics (beans) on Intestinal Functionality: In collaboration with bean breeders from Brazil, effects of soluble bean extracts on intestinal functionality were evaluated by a chicken embryo model, using an intra-amniotic feeding procedure. Bean soluble extracts reduced the relative abundance of potential pathogenic bacterial groups, and upregulated the expression of key intestinal proteins. Soluble extracts from carioca beans may thus improve the intestinal functionality. Effects of Plant origin prebiotics (chia) on Intestinal Functionality, Morphology and Microbiome: Chia is a staple food crop consumed in central and western Latin America. In collaboration with scientists from Vicosa University, Brazil, using a chicken model, soluble extracts from chia seeds improved gut health by reduced abundance of pathogenic bacterial groups, upregulation of key intestinal proteins, and advanced intestinal development.

1. Fast cooking yellow beans provides high bioavailable iron. Bean consumption is often limited, both domestically and abroad, due to long cooking time. In addition, although beans are a major source of dietary iron for many resource-poor populations worldwide where iron deficiency anemia is high, much of that iron is unavailable for absorption. ARS scientists in Ithaca, New York, and East Lansing, Michigan, have now identified specific varieties of yellow beans with more absorbable (bioavailable) iron and cooks in half the time for typical bean varieties. Using the yellow beans, bean growers can produce more nutritious crops that can enhance human health.

2. Novel zinc status nutritional biomarker system. Dietary Zinc deficiency affects twenty percent of the world’s population. There are no reliable zinc biomarkers or animal model in which to test them making the deficiency difficult to detect and monitor. The World’s Health Organization has made zinc deficiency a top priority. An ARS researcher in Ithaca, New York, conducted research that developed a new physiological biomarker of zinc status using red blood cell fatty acids ratio. The zinc biomarker was validated in several human efficacy studies in Benin, West Africa. This accomplishment represents a significant step forward for zinc nutrition.

3. Chicken feeding model for bioactive compounds with nutritional benefits. A reliable method to evaluate components in food that can influence the intestinal microbiome and function, has been lacking. ARS scientist in Ithaca, New York, developed the “in ovo” feeding approach model. This model uses chicken embryo during the last 4-5 days of incubation and immediately after hatching to assess the intestinal microbiome and morphology and to determine uptake of nutrients, and intestinal health. This model could be used to identify positive or negative properties of food extracts, that may influence intestinal development, functionality and nutrient absorption.

Review Publications
Glahn, R.P., Hart, J.J., Beebe, S., Tako, E.N. 2018. Iron bioavailability studies of the first generation of iron-biofortified beans released in Rwanda. In: Tako, E., editor. Fe deficiency, dietary bioavailability and absorption. Basel, Switzerland: MDPI Books. p. 16-27.
Ying, L., Glahn, R.P., Hebb, R., Rizvi, S. 2017. Physico-chemical properties, phytochemicals and DPPH radical scavenging activity of supercritical fluid extruded lentils. LWT - Food Science and Technology. 89:315-321.
Dias, D., Kolba, N.J., Nutti, M., Martino, H., Hart, J.J., Ma, M., Lakshmanan, N., Glahn, R.P., Sha, S., Tako, E.N. 2019. Soluble extracts from carioca beans (Phaseolus vulgaris L.) affect the gut microbiota and iron related brush border membrane protein expression in vivo (Gallus gallus). Food Research International. 123:172-180.
Tako, E. 2017. Intra amniotic administration of raffinose and stachyose affects the intestinal brush border functionality and alters gut microflora populations. In: McCrory, M., editor. Dietary Fibers and Human Health. Basel, Switzerland: MDPI. p. 48-58.
Knez, M., Stangoulis, J., Glibetic, M., Tako, E. 2018. The linoleic acid: dihomo-y-linolenic acid ratio (LA:DGLA)—an emerging biomarker of Zn status. In: Tako, E. Dietary Zn and Human Health. Basel, Switzerland: MDPI Books. p. 1-12.
Guo, Z., Martucci, N., Liu, Y., Mahler, G., Yoo, E., Tako, E.N. 2018. Silicon dioxide nanoparticle exposure affects small intestine function in an in vitro model. Nanotoxicology. 12(5):485-508.