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Hyun-tae Kim,1 John P Loftus,1 Jason W Gagné,1 Michael A Rutzke,2 Raymond P Glahn,3 Joseph J Wakshlag1 1Cornell University College of Veterinary Medicine, Department of Clinical Sciences, Ithaca, NY, USA; 2Cornell University, College of Agriculture and Life Sciences, School of Integrative Plant Sciences, Ithaca, NY, USA; 3Robert W. Holley Center for Agriculture and Health, Agricultural Research Service, USDA, Ithaca, NY, USA Purpose: To evaluate the concentrations of chromium, nickel, molybdenum, silica, and aluminum in several commercially available dry dog foods and compare these with current World Health Organization’s (WHO) mean human daily dietary intake. Conversion of dietary intake per megacalorie (Mcal) for both dog foods and human average intake was performed based on the National Research Council recommendation of a 2,900 kcal diet for comparative purposes to average intake and potential toxic exposure.Materials and methods: Forty-nine over-the-counter dry foods formulated for maintenance of healthy dogs yet listed as all life stage foods were analyzed. Concentrations of the ultra-trace minerals were measured via inductively coupled plasma atomic emission and represented per Mcal for comparative purposes as it relates to common intake in dogs in comparison with humans.Results: Chromium, molybdenum, and aluminum concentrations in all of the dog foods were at levels that would be considered above average human daily consumption on a caloric basis. Nickel and silica calculated intakes per Mcal were comparable with human intake patterns, while both trace minerals displayed outliers exceeding at least twofold of the upper range of human daily intake.Conclusion: Overall, ultra-trace minerals found in dog foods were above the expected average daily intake for humans on a caloric basis. There was no evidence of potential chronic toxic exposure based on presumptive intake extrapolated from WHO published toxic intake concentrations for humans or domestic animals. The large range of silica intake from various foods (2.96–83.67 mg/1,000 kcal) may have health implications in dogs prone to silica urolithiasis. Further studies investigating the bioavailability of these ultra-trace minerals and establishing dietary ultra-trace mineral allowance would be ideal; however, based on these findings, consumption of these ultra-trace minerals in over-the-counter dry dog foods appears safe. Keywords: dog food, ultra-trace minerals, food safety, ICP-AES

Some yellow-colored market classes of dry bean ( Phaseolus vulgaris L.) are valued by consumers as an easy-to-digest, fast cooking alternative to darker colored red and black beans, which in comparison generally have longer cooking times and reduced iron bioavailability. There is evidence that the cooking time of yellow beans is linked to the dietary fiber content and may also contribute to nutrient digestibility and bioavailability. Therefore, 52 fast-, moderate-, and slow-cooking yellow beans with diverse iron bioavailability from five market classes (Amarillo, Canario, Green-yellow, Manteca, and Mayocoba) were selected for total dietary fiber (TDF) analysis. TDF was measured as insoluble (IDF) + soluble (SDF) + oligosaccharides (OLIGO) using method AOAC2011.25. Wide variations in the concentrations of IDF (16.0-23.1%), SDF (1.6-7.7%), OLIGO (1.5-3.4%), and TDF (20.6-31.3%) were detected among the yellow beans with various cooking times. Lower concentrations of IDF in yellow beans were associated with shorter cooking times and higher iron bioavailability. The larger sized Andean yellow beans had more SDF than Middle American. One Mayocoba breeding line from Puerto Rico, PR1146-124, had 42% less OLIGOs than average, and may be useful for breeding low-flatulence beans for consumer acceptability. Fast cooking yellow beans provide the same SDF and OLIGO concentrations as yellow beans with longer cooking times but have the added benefit of shorter cooking times (convenience) and provide more bioavailable iron after cooking., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Karen Cichy reports financial support was provided by 10.13039/100005825National Institute of Food and Agriculture. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

This study aimed to evaluate the effect of extrusion and of open-pan cooking on whole germinated and non-germinated grains of pearl millet (Pennisetum glaucum L. R. Br.), on its chemical-nutritional composition and in vitro iron bioavailability. The experimental design consisted of three flours: non-germination open-pan cooked millet flour (NGOPCMF), germination open-pan cooked millet flour (GOPCMF), and extrusion cooked millet flour (ECMF). The ECMF increased the carbohydrates, iron, manganese, diosmin, and cyanidin and decreased the total dietary fiber, resistant starch, lipids, and total vitamin E, in relation to NGOPCMF. The GOPCMF increased the lysine and vitamin C and decreased the phytate, lipids, total phenolic, total vitamin E, and riboflavin concentration, in relation to NGOPCMF. Furthermore, germinated cooked millet flour and extruded millet flour improved iron availability in vitro compared to non-germinated cooked millet flour. GOPCMF and ECMF generally preserved the chemical-nutritional composition of pearl millet and improved in vitro iron bioavailability; therefore, they are nutritionally equivalent and can be used to develop pearl millet-based products., Competing Interests: Declaration of competing interest The authors affirm that they have no known conflicting financial interests or personal relationships that may have influenced the research presented in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Keigler, Wiesinger, Flint-Garcia and Glahn.)

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., Competing Interests: The authors declare that they have no conflicts of interests., (© 2022 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

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 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McClean, Lee, Howe, Osborne, Grimwood, Levy, Haugrud, Plott, Robinson, Skiba, Tanha, Zamani, Thannhauser, Glahn, Schmutz, Osorno and Miklas.)

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., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Katuuramu, Wiesinger, Luyima, Nkalubo, Glahn and Cichy.)

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 ZnSO 4 .H 2 O 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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

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 ZnSO 4 ·H 2 O (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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Podder, Glahn and Vandenberg.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Broad, Bonneau, Beasley, Roden, Sadowski, Jewell, Brien, Berger, Tako, Glahn, Hellens and Johnson.)

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., (Published by Oxford University Press on behalf of the American Society for Nutrition 2020.)

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 (FeSO 4 ) 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 FeSO 4 , 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 FeSO 4 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 FeSO 4 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 FeSO 4 ( 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 FeSO 4 in the presence of AA and thus provide an excellent source of iron.

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.

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 (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 (FeSO 4 ·7H 2 O (ferrous sulfate hepta-hydrate), FeSO 4 ·H 2 O (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.

Dry beans (Phaseolus vulgaris L.) are a nutrient dense food rich in protein, dietary fiber, minerals, and folate. Consumption of dry beans is relatively low in the United States and this may be due in part to the lack of diversity in bean products beyond whole seeds. Products that incorporate beans in new forms, such as flours, offer the potential to increase dry bean utilization. In this study whole dry beans were milled into flour and used to make gluten free fresh pastas. Six bean varieties each from a different market class (white kidney, navy, otebo, cranberry, dark red kidney and black) were made into pasta. Their consumer appeal was compared to fresh wheat pasta and their nutritional value was compared to fresh wheat pasta and whole boiled beans. The dry bean pastas were nutritionally superior to wheat pasta with higher protein, ash, resistant starch and protein digestibility corrected amino acid score (PDCAAS) as well as lower total starch content. While consumers preferred the flavor, texture and appearance of the wheat pasta to the dry bean pasta, 36% of participants said they would definitely or probably purchase the dry bean pastas from the light colored beans. There was some loss of nutritional value of bean pasta vs. whole boiled beans but this can mostly be attributed to the bean pasta being 90% bean. These results suggest that single variety fresh dry bean pastas have commercial potential in the U.S. as healthy gluten free pasta options.

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 H 2 O; 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 H 2 O). 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bread wheat (Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression (CE) of the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat to up-regulate biosynthesis of two low molecular weight metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - that play key roles in metal transport and nutrition. The CE-OsNAS2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X-ray fluorescence microscopy (XFM) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field-grown CE-OsNAS2 grain and positively correlated with NA and DMA concentrations., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Objective: We collected dietary records over the course of nine months to comprehensively characterize the consumption patterns of Malagasy people living in remote rainforest areas of north-eastern Madagascar., Design: The present study was a prospective longitudinal cohort study to estimate dietary diversity and nutrient intake for a suite of macronutrients, micronutrients and vitamins for 152 randomly selected households in two communities., Setting: Madagascar, with over 25 million people living in an area the size of France, faces a multitude of nutritional challenges. Micronutrient-poor staples, especially rice, roots and tubers, comprise nearly 80 % of the Malagasy diet by weight. The remaining dietary components (including wild foods and animal-source foods) are critical for nutrition. We focus our study in north-eastern Madagascar, characterized by access to rainforest, rice paddies and local agriculture., Participants: We enrolled men, women and children of both sexes and all ages in a randomized sample of households in two communities., Results: Although the Household Dietary Diversity Score and Food Consumption Score reflect high dietary diversity, the Minimum Dietary Diversity-Women indicator suggests poor micronutrient adequacy. The food intake data confirm a mixed nutritional picture. We found that the median individual consumed less than 50 % of his/her age/sex-specific Estimated Average Requirement (EAR) for vitamins A, B12, D and E, and Ca, and less than 100 % of his/her EAR for energy, riboflavin, folate and Na., Conclusions: Malnutrition in remote communities of north-eastern Madagascar is pervasive and multidimensional, indicating an urgent need for comprehensive public health and development interventions focused on providing nutritional security.

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.

Nicotianamine (NA) is a low-molecular weight metal chelator in plants with high affinity for ferrous iron (Fe 2+ ) and other divalent metal cations. In graminaceous plant species, NA serves as the biosynthetic precursor to 2' deoxymugineic acid (DMA), a root-secreted mugineic acid family phytosiderophore that chelates ferric iron (Fe 3+ ) 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. In this study, we utilized the Caco-2 cell system to compare NA and DMA to two known enhancers of Fe bioavailability-epicatechin (Epi) and ascorbic acid (AsA)-and found that both NA and DMA are stronger enhancers of Fe bioavailability than Epi, and NA is a stronger enhancer of Fe bioavailability than AsA. Furthermore, NA reversed Fe uptake inhibition by Myricetin (Myr) more than Epi, highlighting NA as an important target for biofortification strategies aimed at improving Fe bioavailability in staple plant foods.

Biofortification aims to improve the micronutrient concentration and bioavailability in staple food crops. Unlike other strategies utilized to alleviate Fe deficiency, studies of the gut microbiota in the context of Fe biofortification are scarce. In this study, we performed a 6-week feeding trial in Gallus gallus ( n = 15), aimed to investigate the Fe status and the alterations in the gut microbiome following the administration of Fe-biofortified carioca bean based diet (BC) versus a Fe-standard carioca bean based diet (SC). The tested diets were designed based on the Brazilian food consumption survey. Two primary outcomes were observed: (1) a significant increase in total body Hb-Fe values in the group receiving the Fe-biofortified carioca bean based diet; and (2) changes in the gut microbiome composition and function were observed, specifically, significant changes in phylogenetic diversity between treatment groups, as there was increased abundance of bacteria linked to phenolic catabolism, and increased abundance of beneficial SCFA-producing bacteria in the BC group. The BC group also presented a higher intestinal villi height compared to the SC group. Our results demonstrate that the Fe-biofortified carioca bean variety was able to moderately improve Fe status and to positively affect the intestinal functionality and bacterial populations.

The common dry bean ( Phaseolus vulgaris L.) is a nutrient-dense pulse crop that is produced globally for direct human consumption and is an important source of protein and micronutrients for millions of people across Latin America, the Caribbean and Sub-Saharan Africa. Dry beans require large amounts of heat energy and time to cook, which can deter consumers worldwide from using beans. In regions where consumers rely on expensive fuelwood for food preparation, the yellow bean is often marketed as fast cooking. This study evaluated the cooking time and health benefits of five major market classes within the yellow bean seed type (Amarillo, Canary, Manteca, Mayocoba, Njano) over two field seasons. This study shows how the Manteca yellow bean possesses a fast cooking phenotype, which could serve as genetic resource for introducing fast cooking properties into a new generation of dry beans with cooking times <20 min when pre-soaked and <80 min unsoaked. Mineral analysis revealed fast cooking yellow beans have high iron retention (>80%) after boiling. An in vitro digestion/Caco-2 cell culture bioassay revealed a strong negative association between cooking time and iron bioavailability in yellow beans with r values = -0.76 when pre-soaked and -0.64 when unsoaked across the two field seasons. When either pre-soaked or left unsoaked, the highest iron bioavailability scores were measured in the fast cooking Manteca genotypes providing evidence that this yellow market class is worthy of germplasm enhancement through the added benefit of improved iron quality after cooking.

The structure and function of cecal microbiota following the consumption of a zinc (Zn) biofortified wheat diet was evaluated in a well-studied animal model of human nutrition ( Gallus gallus) during a six-week efficacy trial. Using 16S rRNA gene sequencing, a significant increase in β- but not α-microbial diversity was observed in the animals receiving the Zn biofortified wheat diet, relative to the control. No significant taxonomic differences were found between the two groups. Linear discriminant analysis revealed a group of metagenomic biomarkers that delineated the Zn replete versus Zn deficient phenotypes, such that enrichment of lactic acid bacteria and concomitant increases in Zn-dependent bacterial metabolic pathways were observed in the Zn biofortified group, and expansion of mucin-degraders and specific bacterial groups able to participate in maintaining host Zn homeostasis were observed in the control group. Additionally, the Ruminococcus genus appeared to be a key player in delineating the Zn replete microbiota from the control group, as it strongly predicts host Zn adequacy. Our data demonstrate that the gut microbiome associated with Zn biofortified wheat ingestion is unique and may influence host Zn status. Microbiota analysis in biofortification trials represents a crucial area for study as Zn biofortified diets are increasingly delivered on a population-wide scale.

Purpose: To evaluate the concentrations of chromium, nickel, molybdenum, silica, and aluminum in several commercially available dry dog foods and compare these with current World Health Organization's (WHO) mean human daily dietary intake. Conversion of dietary intake per megacalorie (Mcal) for both dog foods and human average intake was performed based on the National Research Council recommendation of a 2,900 kcal diet for comparative purposes to average intake and potential toxic exposure., Materials and Methods: Forty-nine over-the-counter dry foods formulated for maintenance of healthy dogs yet listed as all life stage foods were analyzed. Concentrations of the ultra-trace minerals were measured via inductively coupled plasma atomic emission and represented per Mcal for comparative purposes as it relates to common intake in dogs in comparison with humans., Results: Chromium, molybdenum, and aluminum concentrations in all of the dog foods were at levels that would be considered above average human daily consumption on a caloric basis. Nickel and silica calculated intakes per Mcal were comparable with human intake patterns, while both trace minerals displayed outliers exceeding at least twofold of the upper range of human daily intake., Conclusion: Overall, ultra-trace minerals found in dog foods were above the expected average daily intake for humans on a caloric basis. There was no evidence of potential chronic toxic exposure based on presumptive intake extrapolated from WHO published toxic intake concentrations for humans or domestic animals. The large range of silica intake from various foods (2.96-83.67 mg/1,000 kcal) may have health implications in dogs prone to silica urolithiasis. Further studies investigating the bioavailability of these ultra-trace minerals and establishing dietary ultra-trace mineral allowance would be ideal; however, based on these findings, consumption of these ultra-trace minerals in over-the-counter dry dog foods appears safe., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The amount of Zn absorbed from Zn-biofortified wheat material has been determined using an in vivo model of Zn absorption. The erythrocyte linoleic:dihomo -γ-linolenic acid (LA:DGLA) ratio was used as a biomarker of Zn status. Two groups of chickens (n = 15) were fed different diets: a high-Zn (46.5 μg Zn g -1 ) and a low-Zn wheat-based diet (32.8 μg Zn g -1 ). Dietary Zn intakes, body weight, serum Zn, and the erythrocyte fatty acid profile were measured, and tissues were taken for gene expression analysis. Serum Zn concentrations were greater in the high Zn group (p < 0.05). Duodenal mRNA expression of various Zn transporters demonstrated expression upregulation in the birds fed a low Zn diet (n = 15, p < 0.05). The LA:DGLA ratio was higher in the birds fed the low Zn diet (p < 0.05). The higher amount of Zn in the biofortified wheat resulted in a greater Zn uptake.

Biofortification is a plant breeding method that introduces increased concentrations of minerals in staple food crops (e.g., legumes, cereal grains), and has shown success in alleviating insufficient Fe intake in various human populations. Unlike other strategies utilized to alleviate Fe deficiency, studies of the gut microbiota in the context of Fe biofortification have not yet been reported, although the consumption of Fe biofortified staple food crops has increased significantly over time. Hence, in this study, we performed a 6-week feeding trial in Gallus gallus (n = 14), aimed to investigate the alterations in the gut microbiome following administration of an Fe biofortified bean-based diet (biofortified, BFe) versus a bean based diet with poorly-bioavailable Fe (standard, SFe). Cream seeded carioca bean based diets were designed in an identical fashion to those used in a recent human clinical trial of Fe biofortified beans in Rwanda. We hypothesized that the different dietary Fe contents in the beans based diets will alter the composition and function of the intestinal microbiome. The primary outcomes were changes in the gut microbiome composition and function analyzed by 16S rRNA gene sequencing. We observed no significant changes in phylogenetic diversity between groups. There were significant differences in the composition of the microbiota between groups, with the BFe group harboring fewer taxa participating in bacterial Fe uptake, increased abundance of bacteria involved in phenolic catabolism, and increased abundance of beneficial butyrate-producing bacteria. Additionally, depletion of key bacterial pathways responsible for bacterial viability and Fe uptake suggest that improvements in Fe bioavailability, in addition to increases in Fe-polyphenol and Fe-phytate complexes due to biofortification, led to decreased concentrations of cecal Fe available for bacterial utilization. Our findings demonstrate that Fe biofortification may improve Fe status without negatively altering the structure and function of the gut microbiota, as is observed with other nutritional methods of Fe supplementation. These results may be used to further improve the efficacy and safety of future biofortification efforts in eradicating global Fe deficiency.

Calcium (Ca) is one of the most abundant inorganic elements in the human body and has many important physiological roles. Prebiotics and bioactive peptides are two important substances used to promote calcium uptake. However, the difference in mechanisms of the calcium uptake from these two supplements is not clear. By using the Gallus gallus model and the intra-amniotic administration procedure, the aim of this study was to investigate whether Ca status, intestinal functionality, and health-promoting bacterial populations were affected by prebiotics extracted from chickpea and lentil, and duck egg white peptides (DPs). Eleven groups (non-injected; 18 MΩ H₂O; 4 mmol/L CaCl₂; 50 mg/mL chickpea + 4 mmol/L CaCl₂; 50 mg/mL lentil + 4 mmol/L CaCl₂; 40 mg/mL DPs + 4 mmol/L CaCl₂; 5 mg/mL Val-Ser-Glu-Glu (VSEE) + 4 mmol/L CaCl₂; 50 mg/mL chickpea; 50 mg/mL lentil; 40 mg/mL DPs; 5 mg/mL VSEE) were utilized. Upon hatch, blood, cecum, small intestine, liver and bone were collected for assessment of serum bone alkaline phosphate level (BALP), the relative abundance of intestinal microflora, expression of Ca-related genes, brush border membrane (BBM) functional genes, and liver and bone mineral levels, respectively. The BALP level increased in the presence of lentil, DPs and VSEE ( p < 0.05). The relative abundance of probiotics increased significantly ( p < 0.05) by VSEE + Ca and chickpea. The expression of CalbindinD9k (Ca transporter) increased ( p < 0.05) in Ca, chickpea + Ca and lentil + Ca groups. In addition, the brush border membrane functionality genes expressions increased ( p < 0.05) by the chickpea or lentil extracts. Prebiotics and DPs beneficially affected the intestinal microflora and duodenal villus surface area. This research expands the understanding of the prebiotics' properties of chickpea and lentil extracts, and peptides' effects on calcium metabolism and gut health., Competing Interests: The authors declare no conflict of interest.

Polyphenolic compounds present in the seed coat of common bean (Phaseolus vulgaris L.) are known to act collectively as inhibitors of iron bioavailability. Recent research identified specific polyphenols as being potent Fe uptake inhibitors. That research also identified other polyphenols as being promoters of Fe uptake. The present study extends that work using a Caco-2 cell model to characterize the effects of 43 additional polyphenols on Fe uptake. In addition, this study indicates that the inhibitory compounds have a more potent effect that outweighs the ability of promoting compounds to increase Fe uptake. For example, a ratio of 100:0 epicatechin (a promoter)/myricetin (an inhibitor) produced 78.5 ± 6.7 ng ferritin/mg protein, 90:10 yielded 27.4 ± 3.0, 50:50 yielded 3.42 ± 0.54, and 0:100 yielded 2.26 ± 0.25 ng ferritin/mg protein. A simulation of the relative concentrations of eight major polyphenols (four inhibitors, four promoters) present in a sample of black bean seed coats demonstrated that most of the inhibitory compounds would need to be removed to reduce the negative effect on Fe uptake. In vivo studies are now warranted to confirm the above in vitro effects. Such work would be significant as other bean color classes exist that are likely to have polyphenolic profiles that are more favorable to Fe bioavailability.

This study investigates the effectiveness of two types of prebiotics-stachyose and raffinose-which are present in staple food crops that are widely consumed in regions where dietary Fe deficiency is a health concern. The hypothesis is that these prebiotics will improve Fe status, intestinal functionality, and increase health-promoting bacterial populations in vivo ( Gallus gallus ). By using the intra-amniotic administration procedure, prebiotic treatment solutions were injected in ovo (day 17 of embryonic incubation) with varying concentrations of a 1.0 mL pure raffinose or stachyose in 18 MΩ H₂O. Four treatment groups (50, 100 mg·mL -1 raffinose or stachyose) and two controls (18 MΩ H₂O and non-injected) were utilized. At hatch the cecum, small intestine, liver, and blood were collected for assessment of the relative abundance of the gut microflora, relative expression of Fe-related genes and brush border membrane functional genes, hepatic ferritin levels, and hemoglobin levels, respectively. The prebiotic treatments increased the relative expression of brush border membrane functionality proteins ( p < 0.05), decreased the relative expression of Fe-related proteins ( p < 0.05), and increased villus surface area. Raffinose and stachyose increased the relative abundance of probiotics ( p < 0.05) , and decreased that of pathogenic bacteria. Raffinose and stachyose beneficially affected the gut microflora, Fe bioavailability, and brush border membrane functionality. Our investigations have led to a greater understanding of these prebiotics' effects on intestinal health and mineral metabolism.

Common beans (Phaseolus vulgaris) are the most important legume crops. They represent a major source of micronutrients and a target for essential trace mineral enhancement (i.e. biofortification). To investigate mineral accumulation during seed maturation and to examine whether it is possible to biofortify seeds with multi-micronutrients without affecting mineral bioavailability, three common bean cultivars were treated independently with zinc (Zn) and selenium (Se), the two critical micronutrients that can be effectively enhanced via fertilization. The seed mineral concentrations during seed maturation and the seed Fe bioavailability were analyzed. Common bean seeds were found to respond positively to Zn and Se treatments in accumulating these micronutrients. While the seed pods showed a decrease in Zn and Se along with Fe content during pod development, the seeds maintained relatively constant mineral concentrations during seed maturation. Selenium treatment had minimal effect on the seed accumulation of phytic acid and polyphenols, the compounds affecting Fe bioavailability. Zinc treatment reduced phytic acid level, but did not dramatically affect the concentrations of total polyphenols. Iron bioavailability was found not to be greatly affected in seeds biofortified with Se and Zn. In contrast, the inhibitory polyphenol compounds in the black bean profoundly reduced Fe bioavailability. These results provide valuable information for Se and Zn enhancement in common bean seeds and suggest the possibility to biofortify with these essential nutrients without greatly affecting mineral bioavailability to increase the food quality of common bean seeds., (Published by Elsevier Masson SAS.)

Research methods that predict Fe bioavailability for humans can be extremely useful in evaluating food fortification strategies, developing Fe-biofortified enhanced staple food crops and assessing the Fe bioavailability of meal plans that include such crops. In this review, research from four recent poultry ( Gallus gallus ) feeding trials coupled with in vitro analyses of Fe-biofortified crops will be compared to the parallel human efficacy studies which used the same varieties and harvests of the Fe-biofortified crops. Similar to the human studies, these trials were aimed to assess the potential effects of regular consumption of these enhanced staple crops on maintenance or improvement of iron status. The results demonstrate a strong agreement between the in vitro/in vivo screening approach and the parallel human studies. These observations therefore indicate that the in vitro/Caco-2 cell and Gallus gallus models can be integral tools to develop varieties of staple food crops and predict their effect on iron status in humans. The cost-effectiveness of this approach also means that it can be used to monitor the nutritional stability of the Fe-biofortified crop once a variety has released and integrated into the food system. These screening tools therefore represent a significant advancement to the field for crop development and can be applied to ensure the sustainability of the biofortification approach., Competing Interests: The authors declare no conflict of interest.

Dry beans (Phaseolus vulgaris L.) are a nutrient-dense food rich in protein and micronutrients. Despite their nutritional benefits, long cooking times limit the consumption of dry beans worldwide, especially in nations where fuelwood for cooking is often expensive or scarce. This study evaluated the nutritive value of 12 dry edible bean lines that vary for cooking time (20-89 min) from four market classes (yellow, cranberry, light red kidney, and red mottled) of economic importance in bean-consuming regions of Africa and the Americas. When compared to their slower cooking counterparts within each market class, fast-cooking dry beans retain more protein and minerals while maintaining similar starch and fiber densities when fully cooked. For example, some of the highest protein and mineral retention values were measured in the fast-cooking yellow bean cultivar Cebo Cela, which offered 20% more protein, 10% more iron, and 10% more zinc with each serving when compared with Canario, a slow-cooking yellow bean that requires twice the cooking time to become palatable. A Caco-2 cell culture model also revealed the bioavailability of iron is significantly higher in faster cooking entries (r = -0.537, P = 0.009) as compared to slower cooking entries in the same market class. These findings suggest that fast-cooking bean varieties have improved nutritive value through greater nutrient retention and improved iron bioavailability.

Strategies that enhance the Fe bioavailability of the bean are of keen interest to nutritionists, bean breeders and growers. In beans, the cotyledons contain 75-80% of the total seed Fe, most of which appears to be located within the cotyledon cells. The cotyledon cell walls are known to be resistant to digestion in the stomach and the upper small intestine. Therefore, given the above and the general belief that the primary site for human Fe absorption is the upper small intestine, the present study was designed to determine if the cotyledon cell walls represent a barrier to Fe absorption from the bean. To do so, we utilized high pressure to rupture bean cotyledon cells. The iron bioavailability of cooked bean samples was assessed using an in vitro digestion/Caco-2 cell culture model. Microscopy analyses confirmed that the cotyledon cell walls are highly resistant to pepsin, the low pH of the stomach, and the pancreatic enzymes, indicating that the walls are a barrier to Fe absorption from the bean. Relatively high intracellular pressure (>4000 psi) was required to initiate cell wall rupture. Surprisingly, the lysis of cotyledon cells did not result in a consistent or strong enhancement of bioavailable Fe, suggesting that the liberated intracellular starch and protein influenced the Fe bioavailability by creating a matrix that inhibited the exchange of Fe with the cell transport mechanism. Such observations warrant further pursuit in vivo as the confirmation of these effects would reshape strategies to enhance Fe absorption from beans.

The causes and consequences of geophagy, the craving and consumption of earth, remain enigmatic, despite its recognition as a behavior with public health implications. Iron deficiency has been proposed as both a cause and consequence of geophagy, but methodological limitations have precluded a decisive investigation into this relationship. Here we present a novel in vivo model for assessing the impact of geophagic earth on iron status: Gallus gallus (broiler chicken). For four weeks, animals were gavaged daily with varying dosages of geophagic material or pure clay mineral. Differences in haemoglobin (Hb) across treatment groups were assessed weekly and differences in liver ferritin, liver iron, and gene expression of the iron transporters divalent metal transporter 1 (DMT1), duodenal cytochrome B (DcytB) and ferroportin were assessed at the end of the study. Minimal impact on iron status indicators was observed in all non-control groups, suggesting dosing of geophagic materials may need refining in future studies. However, this model shows clear advantages over prior methods used both in vitro and in humans, and represents an important step in explaining the public health impact of geophagy on iron status.

More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have concentration of approximately 2 μg g(-1) iron (Fe) and 16 μg g(-1) zinc (Zn). The HarvestPlus breeding programs for biofortified rice target 13 μg g(-1) Fe and 28 μg g(-1) Zn to reach approximately 30% of the estimated average requirement (EAR). Reports on engineering Fe content in rice have shown an increase up to 18 μg g(-1) in glasshouse settings; in contrast, under field conditions, 4 μg g(-1) was the highest reported concentration. Here, we report on selected transgenic events, field evaluated in two countries, showing 15 μg g(-1) Fe and 45.7 μg g(-1) Zn in polished grain. Rigorous selection was applied to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different genotype backgrounds.

Zinc (Zn) deficiency is a prevalent micronutrient insufficiency. Although the gut is a vital organ for Zn utilization, and Zn deficiency is associated with impaired intestinal permeability and a global decrease in gastrointestinal health, alterations in the gut microbial ecology of the host under conditions of Zn deficiency have yet to be studied. Using the broiler chicken (Gallus gallus) model, the aim of this study was to characterize distinct cecal microbiota shifts induced by chronic dietary Zn depletion. We demonstrate that Zn deficiency induces significant taxonomic alterations and decreases overall species richness and diversity, establishing a microbial profile resembling that of various other pathological states. Through metagenomic analysis, we show that predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways responsible for macro- and micronutrient uptake are significantly depleted under Zn deficiency; along with concomitant decreases in beneficial short chain fatty acids, such depletions may further preclude optimal host Zn availability. We also identify several candidate microbes that may play a significant role in modulating the bioavailability and utilization of dietary Zn during prolonged deficiency. Our results are the first to characterize a unique and dysbiotic cecal microbiota during Zn deficiency, and provide evidence for such microbial perturbations as potential effectors of the Zn deficient phenotype.

Extrinsic isotopic labeling of food Fe has been used for over 50 years to measure Fe absorption. This method assumes that complete equilibration occurs between the extrinsic and the intrinsic Fe prior to intestinal absorption. The present study tested this assumption via in vitro digestion of varieties of maize, white beans, black beans, red beans, and lentils. Prior to digestion, foods were extrinsically labeled with (58)Fe at concentrations of 1, 10, 50, and 100% of the intrinsic (56)Fe. Following an established in vitro digestion protocol, the digest was centrifuged and the Fe solubilities of the extrinsic (58)Fe and the intrinsic (56)Fe were compared as a measure of extrinsic/intrinsic equilibration. In the beans, significantly more of the extrinsic Fe (up to 2-3 times, p < 0.001) partitioned into the supernatant. The effect varied depending upon the seed coat color, the harvest, and the concentration of the extrinsic Fe. For lentils and maize the extrinsic Fe tended to partition into the insoluble fraction and also varied depending on variety and harvest. There was no crop that consistently demonstrated full equilibration of the extrinsic Fe with the intrinsic Fe. These observations challenge the accuracy of Fe absorption studies in which isotopic extrinsic Fe was used to evaluate Fe absorption and bioavailability.