Genome-wide association and genomic prediction for iron and zinc concentration and iron bioavailability in a collection of yellow dry beans.

Dry bean is a nutrient-dense food targeted in biofortification programs to increase seed iron and zinc levels. The underlying assumption of breeding for higher mineral content is that enhanced iron and zinc levels will deliver health benefits to the consumers of these biofortified foods. This study characterized a diversity panel of 275 genotypes comprising the Yellow Bean Collection (YBC) for seed Fe and Zn concentration, Fe bioavailability (FeBio), and seed yield across 2 years in two field locations. The genetic architecture of each trait was elucidated via genome-wide association studies (GWAS) and the efficacy of genomic prediction (GP) was assessed. Moreover, 82 yellow breeding lines were evaluated for seed Fe and Zn concentrations as well as seed yield, serving as a prediction set for GP models. Large phenotypic variability was identified in all traits evaluated, and variations of up to 2.8 and 13.7-fold were observed for Fe concentration and FeBio, respectively. Prediction accuracies in the YBC ranged from a low of 0.12 for Fe concentration, to a high of 0.72 for FeBio, and an accuracy improvement of 0.03 was observed when a QTN, identified through GWAS, was used as a fixed effect for FeBio. This study provides evidence of the lack of correlation between FeBio estimated in vitro and Fe concentration and highlights the potential of GP in accurately predicting FeBio in yellow beans, offering a cost-effective alternative to the traditional assessment of using Caco2 cell methodologies.

Iron bioavailability of maize (Zea mays L.) after removing the germ fraction.

Maize is a staple food for many communities with high levels of iron deficiency anemia. Enhancing the iron concentrations and iron bioavailability of maize with traditional breeding practices, especially after cooking and processing, could help alleviate iron deficiency in many of these regions. Previous studies on a small number of maize genotypes and maize flour products indicated that degermination (germ fraction removed with processing) could improve the iron bioavailability of maize. This study expanded upon this research by evaluating the iron bioavailability, mineral concentrations, and phytate concentrations of 52 diverse maize genotypes before (whole kernels) and after degermination. Whole and degerminated maize samples were cooked, dried, and milled to produce corn flour. Iron bioavailability was evaluated with an in vitro digestion Caco2 cell bioassay. In 30 of the maize genotypes, bioavailable iron increased when degerminated, thus indicating a higher fractional iron uptake because the iron concentrations decreased by more than 70% after the germ fraction was removed. The remaining 22 genotypes showed no change or a decrease in iron bioavailability after degermination. These results confirm previous research showing that the germ fraction is a strong inhibitory component for many maize varieties. Phytate concentrations in maize flours were greatly reduced with degermination. However, the relationship between phytate concentrations and the iron bioavailability of processed maize flour is complex, acting as either inhibitor or promoter of iron uptake depending on the color of the maize kernels and processing method used to produce flour. Other factors in the maize endosperm fractions are likely involved in the effects of degermination on iron bioavailability, such as vitreous or floury endosperm compositions and the polyphenol content of the bran. This study demonstrates that iron nutrition from maize can be enhanced by selecting genotypes where the inhibitory effect of the bran color and endosperm fraction are relatively low, especially after processing via degermination.

Identifying genes associated with abiotic stress tolerance suitable for CRISPR/Cas9 editing in upland rice cultivars adapted to acid soils.

Five genes of large phenotypic effect known to confer abiotic stress tolerance in rice were selected to characterize allelic variation in commercial Colombian tropical japonica upland rice cultivars adapted to drought-prone acid soil environments (cv. Llanura11 and Porvenir12). Allelic variants of the genes ART1, DRO1, SUB1A, PSTOL1, and SPDT were characterized by PCR and/or Sanger sequencing in the two upland cultivars and compared with the Nipponbare and other reference genomes. Two genes were identified as possible targets for gene editing: SUB1A (Submergence 1A), to improve tolerance to flooding, and SPDT (SULTR3;4) (SULTR-like Phosphorus Distribution Transporter), to improve phosphorus utilization efficiency and grain quality. Based on technical and regulatory considerations, SPDT was targeted for editing. The two upland cultivars were shown to carry the SPDT wild-type (nondesirable) allele based on sequencing, RNA expression, and phenotypic evaluations under hydroponic and greenhouse conditions. A gene deletion was designed using the CRISPR/Cas9 system, and specialized reagents were developed for SPDT editing, including vectors targeting the gene and a protoplast transfection transient assay. The desired edits were confirmed in protoplasts and serve as the basis for ongoing plant transformation experiments aiming to improve the P-use efficiency of upland rice grown in acidic soils.

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability.

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Common Bean V Gene Encodes Flavonoid 3'5' Hydroxylase: A Major Mutational Target for Flavonoid Diversity in Angiosperms.

The classic V (violet, purple) gene of common bean (Phaseolus vulgaris) functions in a complex genetic network that controls seed coat and flower color and flavonoid content. V was cloned to understand its role in the network and the evolution of its orthologs in the Viridiplantae. V mapped genetically to a narrow interval on chromosome Pv06. A candidate gene was selected based on flavonoid analysis and confirmed by recombinational mapping. Protein and domain modeling determined V encodes flavonoid 3'5' hydroxylase (F3'5'H), a P450 enzyme required for the expression of dihydromyricetin-derived flavonoids in the flavonoid pathway. Eight recessive haplotypes, defined by mutations of key functional domains required for P450 activities, evolved independently in the two bean gene pools from a common ancestral gene. V homologs were identified in Viridiplantae orders by functional domain searches. A phylogenetic analysis determined F3'5'H first appeared in the Streptophyta and is present in only 41% of Angiosperm reference genomes. The evolutionarily related flavonoid pathway gene flavonoid 3' hydroxylase (F3'H) is found nearly universally in all Angiosperms. F3'H may be conserved because of its role in abiotic stress, while F3'5'H evolved as a major target gene for the evolution of flower and seed coat color in plants.

Multi-year field evaluation of nicotianamine biofortified bread wheat.

Conventional breeding efforts for iron (Fe) and zinc (Zn) biofortification of bread wheat (Triticum aestivum L.) have been hindered by a lack of genetic variation for these traits and a negative correlation between grain Fe and Zn concentrations and yield. We have employed genetic engineering to constitutively express (CE) the rice (Oryza sativa) nicotianamine synthase 2 (OsNAS2) gene and upregulate biosynthesis of two metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - in bread wheat, resulting in increased Fe and Zn concentrations in wholemeal and white flour. Here we describe multi-location confined field trial (CFT) evaluation of a low-copy transgenic CE-OsNAS2 wheat event (CE-1) over 3 years and demonstrate higher concentrations of NA, DMA, Fe, and Zn in CE-1 wholemeal flour, white flour, and white bread and higher Fe bioavailability in CE-1 white flour relative to a null segregant (NS) control. Multi-environment models of agronomic and grain nutrition traits revealed a negative correlation between grain yield and grain Fe, Zn, and total protein concentrations, yet no correlation between grain yield and grain NA and DMA concentrations. White flour Fe bioavailability was positively correlated with white flour NA concentration, suggesting that NA-chelated Fe should be targeted in wheat Fe biofortification efforts.

Iron Bioavailability from Multiple Biofortified Foods Using an In Vitro Digestion, Caco-2 Assay for Optimizing a Cyclical Menu for a Randomized Efficacy Trial.

BACKGROUND: Inadequate nutritional status contributes to substantial losses in human health and productivity globally. A multiple biofortified food crop trial targeting iron, zinc, and vitamin A deficiencies among young children and their breastfeeding mothers is being conducted in India. OBJECTIVE: We sought to determine the relative iron bioavailability from biofortified and conventional crops and crop combinations representative of a cyclical menu using crops targeted for inclusion in the feeding trial. METHODS: Crops were procured from India, cooked, freeze-dried, and analyzed with an established in vitro digestion/Caco-2 iron bioavailability assay using a fixed sample weight. Crop proportions representative of meals planned for the human study were determined and combined such that samples included either all biofortified or all control crops. Crops were analyzed as single crops (n = 4) or crop combinations (n = 7) by variety (biofortified or control) in triplicate. The primary outcome was iron uptake measured by Caco-2 ferritin production normalized to total Caco-2 protein (nanograms of ferritin/milligrams of cell protein) analyzed for effects of crop variety and crop proportion using generalized linear models. RESULTS: Biofortified pearl millet alone demonstrated higher iron uptake than conventional varieties (5.01 ± 1.66 vs. 2.17 ± 0.96; P = 0.036). Addition of sweet potato or sweet potato + pulse improved iron uptake for all proportions tested in control varieties and select proportions for biofortified varieties (P ≤ 0.05). Two multiple crop combinations demonstrated modestly higher iron uptake from biofortified crops. CONCLUSIONS: Optimizing total iron delivery should consider matrix effects, processing, and promoters/inhibitors of iron absorption in addition to total iron concentration. Future directions include evaluating recipes as prepared for consumption and comparison against human iron bioavailability studies.

Agronomic Performance in Low Phytic Acid Field Peas.

Field pea is a pulse that delivers high protein content, slowly digestible starch and fiber, and many vitamins and minerals, including iron. Naturally occurring plant phytic acid molecules bind iron, lowering its availability for absorption during digestion. Two low phytic acid (lpa) pea lines, 1-2347-144 and 1-150-81, developed by our group had 15% lower yield and 6% lower seed weight relative to their progenitor cultivar. Subsequently, we crossed the two lpa lines and two cultivars, and derived 19 promising lpa pea breeding lines; here we document their agronomic performance based on 10 replicated field trials in Saskatchewan. Seventeen of these lpa lines yielded greater than 95% of the check mean (associated cultivars) and 16 were above 98% of the check mean for 1000 seed weight. The 19 lpa lines showed 27 to 55% lower phytic acid concentration than the check mean. Iron concentrations were similar in all the lpa lines and cultivars, yet the Caco-2 human cell culture assay revealed 14 of the 19 lpa lines had 11 to 55% greater iron bioavailability than check means. Thus, a single round of plant breeding has allowed for closing the gap in performance of low phytic acid pea.

Investigation of Genotype by Environment Interactions for Seed Zinc and Iron Concentration and Iron Bioavailability in Common Bean.

Iron and zinc malnutrition are global public health concerns afflicting mostly infants, children, and women in low- and middle-income countries with widespread consumption of plant-based diets. Common bean is a widely consumed staple crop around the world and is an excellent source of protein, fiber, and minerals including iron and zinc. The development of nutrient-dense common bean varieties that deliver more bioavailable iron and zinc with a high level of trait stability requires a measurement of the contributions from genotype, environment, and genotype by environment interactions. In this research, we investigated the magnitude of genotype by environment interaction for seed zinc and iron concentration and seed iron bioavailability (FeBIO) using a set of nine test genotypes and three farmers' local check varieties. The research germplasm was evaluated for two field seasons across nine on-farm locations in three agro-ecological zones in Uganda. Seed zinc concentration ranged from 18.0 to 42.0 µg g-1 and was largely controlled by genotype, location, and the interaction between location and season [28.0, 26.2, and 14.7% of phenotypic variability explained (PVE), respectively]. Within a genotype, zinc concentration ranged on average 12 µg g-1 across environments. Seed iron concentration varied from 40.7 to 96.7 µg g-1 and was largely controlled by genotype, location, and the interaction between genotype, location, and season (25.7, 17.4, and 13.7% of PVE, respectively). Within a genotype, iron concentration ranged on average 28 µg g-1 across environments. Seed FeBIO ranged from 8 to 116% of Merlin navy control and was largely controlled by genotype (68.3% of PVE). The red mottled genotypes (Rozi Koko and Chijar) accumulated the most seed zinc and iron concentration, while the yellow (Ervilha and Cebo Cela) and white (Blanco Fanesquero) genotypes had the highest seed FeBIO and performed better than the three farmers' local check genotypes (NABE-4, NABE-15, and Masindi yellow). The genotypes with superior and stable trait performance, especially the Manteca seed class which combine high iron and zinc concentrations with high FeBIO, would serve as valuable parental materials for crop improvement breeding programs aimed at enhancing the nutritional value of the common bean.

Genetic control of iron bioavailability is independent from iron concentration in a diverse winter wheat mapping population.

BACKGROUND: Anemia is thought to affect up to 1.6 billion people worldwide. One of the major contributors to low iron (Fe) absorption is a higher proportion of cereals compared to meats and pulse crops in people's diets. This has now become a problem in both the developed and developing world, as a result of both modern food choice and food availability. Bread wheat accounts for 20 % of the calories consumed by humans and is an important source of protein, vitamins and minerals meaning it could be a major vehicle for bringing more bioavailable Fe into the diet. RESULTS: To investigate whether breeding for higher concentrations of Fe in wheat grains could help increase Fe absorption, a multiparent advanced generation intercross (MAGIC) population, encompassing more than 80 % of UK wheat polymorphism, was grown over two seasons in the UK. The population was phenotyped for both Fe concentration and Fe bioavailability using an established Caco-2 cell bioassay. It was found that increasing Fe concentrations in the grains was not correlated with higher Fe bioavailability and that the underlying genetic regions controlling grain Fe concentrations do not co-localise with increased Fe absorption. Furthermore, we show that phytate concentrations do not correlate with Fe bioavailability in our wheat population and thus phytate-binding is insufficient to explain the lack of correlation between Fe bioavailability and Fe concentrations in the wheat grain. Finally, we observed no (Fe bioavailability) or low (Fe concentration) correlation between years for these traits, confirming that both are under strong environmental influence. CONCLUSIONS: This suggests that breeders will have to select not only for Fe concentrations directly in grains, but also increased bioavailability. However the use of numerous controls and replicated trials limits the practicality of adoption of screening by Caco-2 cells by many breeders.

Dual-Fortified Lentil Products-A Sustainable New Approach to Provide Additional Bioavailable Iron and Zinc in Humans.

BACKGROUND: Iron (Fe) and zinc (Zn) deficiencies are global health problems affecting 20% and 33% of the world's population, respectively. Lentil (Lens culinaris Medik.), part of the staple food supply in many countries, can be a potential vehicle for Fe and Zn fortification. OBJECTIVE: We developed a dual-fortification protocol to fortify 3 milled lentil product types (LPTs) [red-football (RF), red-split (RS), and yellow-split (YS)], with NaFeEDTA and ZnSO4.H2O to increase the bioavailable content of Fe and Zn. METHODS: Appropriate Fe and Zn doses were determined to fortify lentils based on RDAs. Relative Fe bioavailability (RFeB%) and phytic acid (PA) content were assessed using an in vitro Caco-2 cell bioassay and PA analysis, respectively. One-factor ANOVA determined the differences in colorimetric score; concentrations of Fe, Zn, and PA; and RFeB% among samples. The least significant difference was calculated with significance level set at P < 0.05. RESULTS: Fe and Zn concentration and RFeB% increased and PA concentration decreased significantly in dual-fortified lentils. Dual-fortified lentil samples had higher RFeB% compared with Fe-fortified (single) samples in all 3 LPTs, whereas RFeB% decreased in Zn-fortified (single) RF and YS samples by 43.4% and 36%, respectively. The RF, RS, and YS samples, fortified with 16 mg Fe and 8 mg Zn/100 g of lentils, provided 27 mg Fe and 14 mg Zn, 28 mg Fe and 13.4 mg Zn, and 29.9 mg Fe and 12.1 mg Zn, respectively. RFeB% of RF, RS, and YS lentil samples increased by 91-307%, 114-522%, and 122-520%, respectively. Again, PA concentrations of RF, RS, and YS lentils were reduced by 0.63-0.53, 0.83-0.71, and 0.96-0.79 mg/g, respectively. CONCLUSIONS: Dual-fortified lentil consumption can cost-effectively provide a significant part of the daily bioavailable Fe and Zn requirements of people with these 2 globally important micronutrient deficiencies.

Effect of Rice GDP-L-Galactose Phosphorylase Constitutive Overexpression on Ascorbate Concentration, Stress Tolerance, and Iron Bioavailability in Rice.

Ascorbate (vitamin C) is an essential multifunctional molecule for both plants and mammals. In plants, ascorbate is the most abundant water-soluble antioxidant that supports stress tolerance. In humans, ascorbate is an essential micronutrient and promotes iron (Fe) absorption in the gut. Engineering crops with increased ascorbate levels have the potential to improve both crop stress tolerance and human health. Here, rice (Oryza sativa L.) plants were engineered to constitutively overexpress the rice GDP-L-galactose phosphorylase coding sequence (35S-OsGGP), which encodes the rate-limiting enzymatic step of the L-galactose pathway. Ascorbate concentrations were negligible in both null segregant (NS) and 35S-OsGGP brown rice (BR, unpolished grain), but significantly increased in 35S-OsGGP germinated brown rice (GBR) relative to NS. Foliar ascorbate concentrations were significantly increased in 35S-OsGGP plants in the vegetative growth phase relative to NS, but significantly reduced at the reproductive growth phase and were associated with reduced OsGGP transcript levels. The 35S-OsGGP plants did not display altered salt tolerance at the vegetative growth phase despite having elevated ascorbate concentrations. Ascorbate concentrations were positively correlated with ferritin concentrations in Caco-2 cells - an accurate predictor of Fe bioavailability in human digestion - exposed to in vitro digests of NS and 35S-OsGGP BR and GBR samples.

Impact of Ascorbic Acid on the In Vitro Iron Bioavailability of a Casein-Based Iron Fortificant.

A new iron-casein complex (ICC) has been developed for iron (Fe) fortification of dairy matrices. The objective was to assess the impact of ascorbic acid (AA) on its in vitro bioavailability in comparison with ferrous sulfate (FeSO4) and ferric pyrophosphate (FePP). A simulated digestion coupled with the Caco-2 cell culture model was used in parallel with solubility and dissociation tests. Under diluted acidic conditions, the ICC was as soluble as FeSO4, but only part of the iron was found to dissociate from the caseins, indicating that the ICC was an iron chelate. The Caco-2 cell results in milk showed that the addition of AA (2:1 molar ratio) enhanced iron uptake from the ICCs and FeSO4 to a similar level (p = 0.582; p = 0.852) and to a significantly higher level than that from FePP (p < 0.01). This translated into a relative in vitro bioavailability to FeSO4 of 36% for FePP and 114 and 104% for the two ICCs. Similar results were obtained from water. Increasing the AA to iron molar ratio (4:1 molar ratio) had no additional effect on the ICCs and FePP. However, ICC absorption remained similar to that from FeSO4 (p = 0.666; p = 0.113), and was still significantly higher than that from FePP (p < 0.003). Therefore, even though iron from ICC does not fully dissociate under gastric digestion, iron uptake suggested that ICCs are absorbed to a similar amount as FeSO4 in the presence of AA and thus provide an excellent source of iron.

Iron Concentrations in Biofortified Beans and Nonbiofortified Marketplace Varieties in East Africa Are Similar.

BACKGROUND: The predominant bean iron (Fe) biofortification approach is to breed for high Fe concentration and assumes the average Fe concentration is 50 µg/g. This approach also assumes that a 40 µg/g increase is sustainable and Fe bioavailability will not decrease to negate the increase in Fe. OBJECTIVE: The overall objective was to determine if bean Fe biofortification via breeding for high Fe is producing beans with higher Fe concentration relative to nonbiofortified lines found in the East Africa marketplace. METHODS: Seventy-six marketplace samples (East Africa Marketplace Collection; EAMC), and 154 genotypes known to be representative of the marketplace were collected from breeders in the Pan-Africa Bean Research Alliance (designated the East Africa Breeder Collection; EABC). Within the EAMC and EABC were 18 and 35 samples, respectively, that were released as biofortified lines. All samples were measured for Fe concentration. The Caco-2 cell bioassay assessed Fe bioavailability of the EAMC. Biofortified versus nonbiofortified samples were compared by the appropriate t-test or ANOVA. RESULTS: The Fe concentration of the 58 nonbiofortified EAMC lines was (mean ± SD [range]) 71 ± 9 µg/g (52-93 µg/g) which did not differ significantly from the 18 biofortified EAMC varieties (71 ± 11 µg/g [55-94 µg/g]). The Fe concentration of the 119 nonbiofortified EABC varieties was 66 ± 7 µg/g (51-90 µg/g) which was significantly different (P < 0.0001) from the 35 EABC biofortified lines (73 ± 9 µg/g [60-91 µg/g]). However, the EABC biofortified lines were not different from the nonbiofortified EAMC samples. In the Caco-2 cell bioassay, biofortified EAMC varieties did not deliver more Fe compared with nonbiofortified EAMC varieties. CONCLUSIONS: The assumptions of the high Fe bean biofortification approach are not met in the East African marketplace. Iron concentration and bioavailability measurement indicate the biofortified bean varieties are providing no additional dietary Fe.

Nicotianamine-chelated iron positively affects iron status, intestinal morphology and microbial populations in vivo (Gallus gallus).

Wheat flour iron (Fe) fortification is mandatory in 75 countries worldwide yet many Fe fortificants, such as Fe-ethylenediaminetetraacetate (EDTA), result in unwanted sensory properties and/or gastrointestinal dysfunction and dysbiosis. Nicotianamine (NA) is a natural chelator of Fe, zinc (Zn) and other metals in higher plants and NA-chelated Fe is highly bioavailable in vitro. In graminaceous plants NA serves as the biosynthetic precursor to 2' -deoxymugineic acid (DMA), a related Fe chelator and enhancer of Fe bioavailability, and increased NA/DMA biosynthesis has proved an effective Fe biofortification strategy in several cereal crops. Here we utilized the chicken (Gallus gallus) model to investigate impacts of NA-chelated Fe on Fe status and gastrointestinal health when delivered to chickens through intraamniotic administration (short-term exposure) or over a period of six weeks as part of a biofortified wheat diet containing increased NA, Fe, Zn and DMA (long-term exposure). Striking similarities in host Fe status, intestinal functionality and gut microbiome were observed between the short-term and long-term treatments, suggesting that the effects were largely if not entirely due to consumption of NA-chelated Fe. These results provide strong support for wheat with increased NA-chelated Fe as an effective biofortification strategy and uncover novel impacts of NA-chelated Fe on gastrointestinal health and functionality.

Iron- and Zinc-Fortified Lentil (Lens culinaris Medik.) Demonstrate Enhanced and Stable Iron Bioavailability After Storage.

Lentil (Lens culinaris Medik.) is a quick-cooking, rapidly expanding protein-rich crop with high iron (Fe) and zinc (Zn), but low bioavailability due to the presence of phytate, similar to other grains. Lentils dual fortified with Fe and Zn can significantly improve the bioavailable Fe and Zn content. Three milled lentil product types (LPTs) were fortified with Fe using NaFeEDTA [ethylenediaminetetraacetic acid iron (III) sodium salt] (Fe fortified) or Zn from ZnSO4·H2O (Zn fortified), or both (dual fortified). Fe, Zn, phytic acid (PA) concentration, and relative Fe bioavailability (RFeB%) were assessed for samples from two fortified batches (initial and for 1 year stored). Fe, Zn, and RFeB% increased significantly in two batches of samples from the three LPTs, and decreased by 5-15% after 1 year of storage. PA concentration decreased from 8 to 15% after fortification of all samples from two batches of the three LPTs but showed different patterns of influence after storage. Dual-fortified lentil fortified with 24 mg Fe and 12 mg Zn 100 g-1 lentil had the highest amount of Fe and Zn, and the lowest PA concentration, and RFeB% was increased from 91.3 to 519.5%. Significant (p ≤ 0.01) Pearson correlations were observed between Fe concentration vs. PA:Fe molar ratio (MR), Fe concentration vs. RFeB%, RFeB% vs. PA:Fe MR, and Zn concentration vs. PA:Zn MR in all samples from two batches of the three LPTs. In conclusion, dual-fortified lentil can contribute significant bioavailable Fe and Zn to populations at risk of Fe and Zn deficiency.

Polyphenolic Profiles of Yellow Bean Seed Coats and Their Relationship with Iron Bioavailability.

Previous work with Caco-2 cell cultures has shown that individual polyphenols can either promote or inhibit iron uptake. This investigation was designed to characterize the relationship between iron bioavailability and seed coat polyphenol composition in a panel of 14 yellow beans representing five market classes with the potential for fast cooking time and high iron content. The study included two white and two red mottled bean lines, which represent high and low iron bioavailability capacity in dry beans, respectively. Polyphenols were measured quantitatively by high-performance liquid chromatography-mass spectrometry (HPLC-MS)/UV and iron bioavailability of seed coat extracts was measured in Caco-2 assays. Thirteen of the yellow bean seed types contained high concentrations (up to 35.3 ± 2.7 µmol/g) of kaempferol 3-glucoside (k 3-g), a known promoter of iron uptake. A general association between the ratio of promoting to inhibiting polyphenols (P/I) and iron uptake was observed. The presence of iron uptake inhibiting condensed tannins proportionately countered the promotional effects of kaempferol compounds. Unidentified factors present in seed coats other than polyphenols also appeared to affect iron uptake.

Iron Fortification and Bioavailability of Chickpea (Cicer arietinum L.) Seeds and Flour.

Iron (Fe) deficiency is one of the most common nutritional disorders, and is mainly due to insufficient intake of bioavailable Fe. Chickpea (Cicer arietinum L.) was examined as a potential vehicle for Fe fortification. Fortificants (FeSO4·7H2O (ferrous sulfate hepta-hydrate), FeSO4·H2O (ferrous sulfate mono-hydrate) and NaFeEDTA (ethylenediaminetetraacetic acid iron (iii) sodium salt)) were applied by a spraying and drying method. At 2000 µg g-1 iron fortificant, the fortified split desi seeds (dal), desi flour and kabuli flour supplied 18-19 mg, 16-20 mg and 11-19 mg Fe per 100 g, respectively. The overall consumer acceptability using a nine-point hedonic scale for sensory evaluation demonstrated that NaFeEDTA-fortified cooked chickpea (soup and chapatti) scored the highest among the three fortificants. Lightness (L*), redness (a*) and yellowness (b*) of Fe-fortified products changed over time. However, no organoleptic changes occurred. Fe bioavailability was increased by 5.8-10.5, 15.3-25.0 and 4.8-9.0 ng ferritin mg-1 protein for cooked split desi seeds (soup), desi chapatti and kabuli chapatti, respectively, when prepared using Fe-fortified chickpea. Desi chapatti showed significantly higher Fe bioavailability than the other two. The increase in Fe concentration and bioavailability in fortified chickpea products demonstrated that these products could provide a significant proportion of the recommended daily Fe requirement.

An In Vivo (Gallus gallus) Feeding Trial Demonstrating the Enhanced Iron Bioavailability Properties of the Fast Cooking Manteca Yellow Bean (Phaseolus vulgaris L.).

The common dry bean (Phaseolus vulgaris L.) is a globally produced pulse crop and an important source of micronutrients for millions of people across Latin America and Africa. Many of the preferred black and red seed types in these regions have seed coat polyphenols that inhibit the absorption of iron. Yellow beans are distinct from other market classes because they accumulate the antioxidant kaempferol 3-glucoside in their seed coats. 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. Hence, this study compared the iron bioavailability of three faster cooking yellow beans with contrasting seed coat colors from Africa (Manteca, Amarillo, and Njano) to slower cooking white and red kidney commercial varieties. Iron status and iron bioavailability was assessed by the capacity of a bean based diet to generate and maintain total body hemoglobin iron (Hb-Fe) during a 6 week in vivo (Gallus gallus) feeding trial. Over the course of the experiment, animals fed yellow bean diets had significantly (p ≤ 0.05) higher Hb-Fe than animals fed the white or red kidney bean diet. This study shows that the Manteca yellow bean possess a rare combination of biochemical traits that result in faster cooking times and improved iron bioavailability. The Manteca yellow bean is worthy of germplasm enhancement to address iron deficiency in regions where beans are consumed as a dietary staple.

Soluble extracts from carioca beans (Phaseolus vulgaris L.) affect the gut microbiota and iron related brush border membrane protein expression in vivo (Gallus gallus).

The effect of soluble extracts with putative prebiotic ability extracted from various bean varieties on the intestinal brush border membrane (BBM) iron related proteins, and intestinal bacterial populations were evaluated using the Gallus gallus model and by the intra-amniotic administration procedure. Eight treatment groups [(non-injected; 18 MΩ H2O; 40 mg/mL Inulin; 50 mg/mL BRS Perola (carioca standard); 50 mg/mL BRS Cometa (carioca, Fe biofortified); 50 mg/mL BRS Esteio (black, standard); 50 mg/mL SMN 39 (black, Fe biofortified); 50 mg/mL BRS Artico (white, standard)] were utilized. Tested groups reduced the relative abundance of Clostridium and E. coli compared to the Inulin group (positive control) and they did not affect the relative abundance of Bifidobacterium and Lactobacillus compared to the negative control (18MΩ H2O). The relative expression of zinc transporter 1, ferroportin and amino peptidase were up-regulated in the BRS Cometa group (Fe-biofortified carioca beans). Results suggest that soluble extracts from carioca beans may improve the iron bioavailability by affecting intestinal bacterial populations, and BBM functionality.