Effect of Iron Source on Color and Appearance of Micronutrient‐Fortified Corn Flour Tortillas

Iron deficiency anemia is a widespread occurrence. Consequently, iron is commonly added in cereal fortification programs. However, many iron sources cause undesirable sensory changes, especially color changes, in the food being fortified. This study evaluated the effect of different iron sources on CIE L*a*b* color values and sensory color perception in fortified corn tortillas. Corn masa flour was fortified with micronutrient premix containing vitamins, zinc, and one of eight iron compounds. Iron sources included ferrous fumarate (F), ferrous sulfate (S), ferric orthophosphate (OP), ferrous lactate (L), ferrous gluconate (G), ferric pyrophosphate (PP), sodium iron (III)‐EDTA, and A‐131 electrolytic iron (E), with addition levels adjusted based on bioavailability. Control (Ct) samples were prepared with all micronutrients except iron. All iron‐fortified tortillas had lower L* values and were significantly darker than control tortillas. Based on instrumental color values and Mexican regulatory recommendations, five treatments were selected for further testing. A difference‐from‐control sensory test was conducted comparing PP, E, OP, F, and S with Ct tortillas. Sensory rankings were C t > E = PP > OP > F > S. A‐131 electrolytic iron is recommended for fortification of corn tortillas due to minimal effect on color and significantly lower cost than other iron sources evaluated.     

Inhibition of iron uptake from iron salts and chelates by divalent metal cations in intestinal epithelial cells

Iron chelates, namely, ferrous bisglycinate and ferric EDTA, are promising alternatives to iron salts for food fortification. The objectives of this study were to compare iron uptake from radiolabeled ferrous sulfate, ferrous ascorbate, ferrous bisglycinate, ferric chloride, ferric citrate, and ferric EDTA by Caco-2 cells with different iron status and in the presence of divalent metal cations. Iron-loaded Caco-2 cells, with reduced DMT-1 and elevated HFE mRNA levels, down-regulated uptake from ferrous ascorbate and bisglycinate but not from ferric compounds. Nevertheless, iron uptake from all compounds was markedly inhibited in the presence of 100-fold molar excess of Co2+ and Mn2+ cations, with ferrous compounds showing a greater percent reduction. Our results suggest that ferrous iron is the predominant form of iron taken up by intestinal epithelial cells and the DMT-1 pathway is the major pathway for uptake. Iron uptake from chelates appears to follow the same pathway as uptake from salts.     

Iron availability from iron-fortified spirulina by an in vitro digestion/Caco-2 cell culture model

Iron deficiency, one of the most important nutritional problems in the world, can be caused not only by foods deficient in iron but also by poor availability of dietary iron. Iron food fortification in combination with highly available iron from supplements could effectively reduce this deficiency. The aim of this study was to examine the iron availability from iron-fortified spirulina. We have used an in vitro digestion/Caco-2 cell culture system to measure iron spirulina availability and made a comparison with those of beef, yeast, wheat floor, and iron sulfate plus ascorbic acid as a reference. Iron availability was assessed by ferritin formation in Caco-2 cells exposed to digests containing the same amount of iron. Our results demonstrate a 27% higher ferritin formation from beef and spirulina digests than from digests of yeast and wheat flour. When iron availability was expressed per microgram of iron used in each digest, a 6.5-fold increase appeared using spirulina digest in comparison with meat. In view of this observed high iron availability from spirulina, we conclude that spirulina could represent an adequate source of iron.     

Bioavailability of iron from wheat aegilops derivatives selected for high grain iron and protein contents

A coupled in vitro digestion/Caco-2 model was employed to assess iron bioavailability from wheat Aegilops derivatives selected for high iron and protein contents. The iron content in wheat genotypes used in this study correlated to a great extent with both protein (r = 0.80) and phytate (r = 0.68) contents. The iron bioavailability was based on Caco-2 cell ferritin formation from cooked digests of these derivatives (relative to WL711 control) and correlated positively with dialyzable iron (r = 0.63) and total iron content (r = 0.38) but not with the phytate content. The apparently decreased phytate/iron molar ratios, however, correlated negatively (r = -0.42) with the iron bioavailability, justifying the utilization of these parameters in biofortification programs. Iron bioavailability in the derivatives increased up to 1.5-fold, corresponding to a 1.5-2.2-fold increase observed in iron content over control. These data suggest that biofortification for iron proportionately leading to higher iron bioavailability will be the most feasible and cost-effective approach to combat micronutrient deficiency.     

Mineral dialyzability in milk and fermented dairy products fortified with FeNaEDTA

Iron, zinc, and calcium dialyzability and ascorbic acid (AA) concentrations were evaluated in milk and yogurt fortified with FeNaEDTA (FE) or ferrous sulfate (FS) as a control, with or without AA addition. The values obtained for FE iron dialyzability in milk were much higher than those obtained for FS. The addition of AA to milk improved Fe dialyzability when using FS and slightly decreased Fe dialyzability in the FE-fortified nonfermented samples. Milk fermentation increased iron availability from both iron sources. Zinc and calcium dialyzability in products containing any of the two iron sources was increased in fermented milks. EDTA improved Zn dialyzability from intrinsic zinc in every manufactured dairy product. Whereas for milks fortified with FS and stored at 4 degrees C for 24 h, the AA content remained close to the original concentration, a higher AA degradation was observed when milks were fortified with FE.     

IRON IS REQUIRED FOR THE INDUCTION OF ROOT FERRIC CHELATE REDUCTASE ACTIVITY IN IRON-DEFICIENT TOMATO

Two mutants of tomato and their corresponding wild-type genotypes, Tfer/TFER and chloronerva/Bonner Beste, were grown in nutrient solution under conditions leading to iron (Fe) deficiency. Iron deficiency caused decreases in growth, leaf chlorosis, and changes in the morphology of roots. Ferric chelate reductase activities of whole roots were generally lower in Fe-deficient plants than in control, Fe-sufficient plants. Plants grown for 7 days without Fe, however, had transient increases in whole root ferric chelate reductase activity after the addition of small amounts of Fe (2 μM) to the nutrient solution. Also, adding sequential 0.5 μM Fe pulses to the nutrient solution led to high whole root ferric chelate reductase activities. Similar results were obtained with a protocol using excised root tips instead of whole root systems to measure ferric chelate reductase activities. The protocol using root tips generally gave higher ferric chelate reductase rates than the method using whole roots, due to the localized expression of the enzyme in the distal root zones.     

Effect of bread baking on the bioavailability of hydrogen-reduced iron powder added to unenriched refined wheat flour

Elemental iron powders are widely used to fortify flour and other cereal products. Our objective was to test the hypothesis that baking enhances the bioavailability of elemental iron powders by oxidizing Fe(0) to Fe(2+) or Fe(3+). An in vitro digestion/Caco-2 cell culture model and a piglet model were used to measure bioavailability. Bread flour, either unfortified or fortified with hydrogen-reduced (HR) iron powder or FeSO(4) (300 mg Fe/kg flour), was baked into bread. For the in vitro studies, bread samples were treated with pepsin at pH 2, 3, 4, 5, 6, or 7 and subsequently incubated with pancreatic enzymes at pH 7 in a chamber positioned above monolayers of cultured Caco-2 cells. Ferritin formation in the cells was used as an index of iron bioavailability. Ferritin formation in cells fed HR Fe bread was similar to cells fed FeSO(4) bread when the peptic digestion was conducted at a pH 2 but lower when the peptic phase was conducted at pH 3, 4, 5, 6, or 7 (P < 0.05). Pig diets containing 35% dried bread were prepared and fed to cross-bred (Hampshire x Landrace x Yorkshire) anemic pigs in two studies. The rate of increase in hemoglobin Fe over the feeding period was used to calculate relative biological value (RBV), an index of iron bioavailability. In the first pig study, RBV of HR Fe added to flour prior to baking was 47.9% when compared to FeSO(4) fortified flour (P < 0.05). In the second pig study, a third treatment consisting of unfortified bread with HR iron added during diet mixing (after bread baking) was included. RBVs of the HR Fe diet (Fe added after baking) and HR Fe diet (Fe added before baking) were 40.1% and 53.5%, respectively, compared to the FeSO(4) diet. Differences in RBV between the HR Fe (before and after baking) and FeSO(4) (before baking) treatment groups were significant, but the difference between the before and after HR treatment groups was not significant. We conclude that bread baking does not enhance the bioavailability of elemental iron powders.     

Uptake and translocation of iron from ferrated rhodotorulic acid in tomato

Micro‐organisms may develop an iron‐deficiency stress when grown in an alkaline environment and secrete ferric‐specific chelators known as siderophores. Some of these siderophores may have stability constants which can exceed 30. This is comparable to the synethetic Fe chelate FeEDDHA. Our objective was to determine if the Fe‐efficient T3238 FER tomato and the Fe‐inefficient T3238 fer tomato could use iron supplied as the siderophore ferrated‐rhodotorulic acid. After these two tomato cultivars were grown with adequate nutrition to obtain plants large enough for experimental testing, they were grown without iron until Fe‐deficiency‐stress symptoms developed and then iron was supplied as ferrated‐rhodoturulic acid. Iron efficient T3238 FER tomato utilized iron supplied as the siderophore and greened whereas, the Fe‐inefficient T3238 fer tomato plants were chlorotic because they could not use the iron in the siderophore. This study demonstrated that some higher plants subjected to various degrees of iron‐deficiency stress in nutrient culture may derive their iron requirement from siderophores of microbial origin.     

Biofortification of common bean as a complementary approach to addressing zinc deficiency in South Africans

Deficiencies of vitamin A, iodine, iron and zinc (Zn) in humans are caused partly by the consumption of food that has insufficient quantities of these. Their deficiency has a negative impact on the health, wellbeing, social and economic status of human beings. A national survey conducted in 2012 identified deficiencies of vitamin A, Fe, and Zn among other nutrients in South Africans and regarded the deficiencies of vitamin A and Fe as a moderate but not Zn. This review discusses causes of Zn prevalence in low-income South Africans and that it is largely caused by the low content of Zn in their diets. Initiatives to reduce Zn deficiency include fortification of wheat products and maize meal which has failed to address it successfully. Weaknesses of fortification include high cost of fortified food products to low-income populations, poor regulation in ensuring compliance in fortification, non-fortification of sorghum meal, and leaching of fortified nutrients during processing. This review suggests Zn-biofortification of locally-preferred common bean cultivars as an alternative strategy to compliment fortification. The review also discusses advantages of adopting biofortified Nutritional Andean common beans. Furthermore, the review suggests initiatives including evaluation of the common bean genotypes’ adaptation to different agro-ecologies.     

Iron uptake by Caco-2 cells from NaFeEDTA and FeSO4: Effects of ascorbic acid, pH, and a Fe(II) chelating agent

Sodium iron(III) ethylenediaminetetraacetate (NaFeEDTA) has considerable promise as an iron fortificant because of its high bioavailability in foods containing iron absorption inhibitors. In this study, uptakes of iron from NaFeEDTA, FeSO4, and FeCl3 by Caco-2 cells were compared in the absence or presence of ascorbic acid (AA), an iron absorption enhancer; at selected pH levels; and in the absence or presence of an iron absorption inhibitor, bathophenanthroline disulfonic acid (BPDS). Ferritin formation in the cells was used as the indicator of iron uptake. Uptake from all three Fe sources was similar in the absence of AA. Adding AA at a 5:1 molar excess as compared to Fe increased uptake by 5.4-, 5.1-, and 2.8-fold for FeSO4, FeCl3, and NaFeEDTA, respectively. The smaller effect of AA on uptake from NaFeEDTA may be related to the higher solubility of NaFeEDTA and/or the strong binding affinity of EDTA for Fe3+, which may prevent AA and duodenal cytochrome b from effectively reducing EDTA-bound Fe. Uptake was inversely related to the pH of the media over a range of 5.8-7.2. Because uptake by DMT-1 is proton-coupled, the inverse relationship between pH and Fe uptake in all three iron sources suggests that they all follow the DMT-1 pathway into the cell. Adding BPDS to the media inhibited uptake from all three iron compounds equally. Because BPDS binds Fe2+ but not Fe3+ and because only Fe2+ is transported by DMT-1, the finding that BPDS inhibited uptake from NaFeEDTA suggests that at least some iron dissociates from EDTA and is reduced just as simple inorganic iron at the brush border membrane of the enterocyte. Taken together, these results suggest that uptake of iron from NaFeEDTA by intestinal enterocytes is regulated similarly to uptake from iron salts.     

Tissue iron distribution and urinary mineral excretion vary depending on the form of iron (FeSO4 or NaFeEDTA) and the route of administration (oral or subcutaneous) in rats given high doses of iron

Sodium iron ethylenediaminetetraacetate (NaFeEDTA) has considerable promise as an iron fortificant in food. However, effects of administering high levels of NaFeEDTA on tissue iron distribution and mineral excretion are not well understood. The objectives of this study were to assess nonheme iron distribution in the body and urinary excretion of Ca, Mg, Cu, Fe, and Zn after daily administration of high levels of iron to rats over 21 days. Iron was either given orally with food or injected subcutaneously, as either FeSO 4 or NaFeEDTA. Selected tissues were collected for nonheme iron analysis. Estimated total body nonheme iron levels were similar in rats fed NaFeEDTA or FeSO 4, but the tissue distribution was different: it was 53% lower in the liver and 86% higher in the kidneys among rats fed NaFeEDTA than among those fed FeSO 4. In contrast, body nonheme iron was 3.2-fold higher in rats injected with FeSO 4 than in rats injected with NaFeEDTA. Administering NaFeEDTA orally elevated urinary Cu, Fe, and Zn excretion compared with FeSO 4 (1.41-, 11.9-, and 13.9-fold higher, respectively). We conclude that iron is dissociated from the EDTA complex prior to or during intestinal absorption. A portion of intact FeEDTA may be absorbed via a paracellular route at high levels of intake but is mostly excreted in the urine. Metal-free EDTA may be absorbed and cause elevated urinary excretion of Fe, Cu, and Zn.     

Iron deficiency in wheat grown on the southern plains

Extensive areas of chlorotic winter wheat (Triticum aestivun L.) were observed on calcareous soils in western Oklahoma. Spraying severely chlorotic wheat with ferrous sulfate (FeSO₄) resulted in greening within a week and doubled herbage yield a month later. Intensive grazing of wheat prior to jointing induced no to severe chlorosis in 33 wheat cultivars tested over three years. After peak intensity in early April, the chlorosis diminished and was not visible when the wheat was fully headed in May. Overall, we believe increased iron (Fe) deficiency in wheat on the Southern Plains is due to increased use of Fe‐inefficient cultivars and stress induced by grazing. Wheat cultivars less susceptible to Fe deficiency are commercially available.     

Perspectives of folate biofortification of cereal grains

One sixth of the world’s population is suffering from hidden hunger that indicates a gross malnutrition particularly among children and women of third world countries. The deficiency of micro nutrients, especially iron (Fe) causes a number of ailments such as megaloblastic anemia and neural tube defects in poor population. There is a dire need to supplement iron in the diet. Current efforts implicate fortification of wheat flour and other grains with different iron formulations such as ethylenediaminetetraacetic acid (EDTA), FeSO₄ and elemental iron. However, all such interventions are not sustainable due to logistic and quality assurance problems in resource-limited settings. For a long term solution, development of crop plants with increased micronutrients and iron bioavailability is essential. Therefore, biofortification of cereal grains using translational genomics approaches for enhancement of folate through genome editing in cereals is inevitable to mitigate the folate deficiency in poor remote population in a cost effective manner.     

Fortification of milk with calcium: effect on calcium bioavailability and interactions with iron and zinc

Calcium solubility, dialysability, and transport and uptake (retention + transport) by Caco-2 cells as indicators of calcium bioavailability have been estimated in the in vitro gastrointestinal digests of milk and calcium fortified milk. A significant linear correlation (p < 0.05) was obtained between calcium uptake and the amount of soluble calcium added to the cells, and also between percentage calcium uptake and the calcium measured in the analyzed samples. The solubility, dialysis, transport, and uptake values are higher (p < 0.05) for calcium fortified milks than for nonfortified milks; that is, calcium fortification increases not only calcium content but also its bioavailability. An inhibitory effect of calcium from fortified milks upon iron absorption was found. The observed effect of calcium from fortified milks upon zinc bioavailability depends on the in vitro method used, zinc solubility and dialysis decrease in calcium fortified milks, and percentage zinc uptake remains unchanged.     

Induction of in vivo root ferric chelate reductase activity in fruit tree rootstock

A method has been developed to consistently induce increases in root ferric chelate reductase activity in the fruit tree rootstock GF 677 (Prunus amygdalopersica) grown under iron (Fe) deficiency. Clonal GF 677 plants were grown hydroponically in a growth chamber with 0 or 90 μM Fe(III)‐EDTA. Root ferric chelate reductase activity was measured in vivo using BPDS. Plants grown without Fe developed visible symptoms of chlorosis and had lower root ferric chelate reductase activities than those grown with Fe. Root ferric chelate reductase activities were 0.1–1.9 and 0.6–5.3 nmol of Fe reduced per gram of fresh mass and minute, respectively, in Fe‐deficient and sufficient plants. However, when plants grown without Fe for several days were resupplied with 180 μM of Fe(III)‐EDTA, FC‐R activities increased within 1 day. The FC‐R values after Fe resupply were 20‐fold higher than those found in Fe‐deficient plants and 5‐fold higher than those found in the Fe‐sufficient controls. After three days of the Fe treatments the FC‐R activities had decreased again to the control values. The reduction of Fe was localized at the subapical root zone. In the conditions used we have found no decreases of the nutrient solution pH values, indicating that this type of response is not strong enough to be detected in peach tree rootstocks. Also, no major changes in root morphology have been found in response to Fe deficiency. This ferric chelate reductase induction protocol may be used in screening assays to select rootstock genotypes tolerant to Fe chlorosis.     

Tissue iron distribution and adaptation of iron absorption in rats exposed to a high dietary level of NaFeEDTA

Although it has been shown that iron absorption from NaFeEDTA, a promising iron fortificant, is effectively down-regulated in iron-loaded rats, effects of prolonged exposure to high dietary levels of NaFeEDTA are not well understood. The objectives of this study were to determine whether rats can adapt to a high dietary level of NaFeEDTA by down-regulating iron absorption, and to determine effects on tissue iron distribution, with or without an iron absorption inhibitor. Male Sprague-Dawley rats were exposed to diets supplemented with FeSO4 or NaFeEDTA at 1200 mg of Fe/kg of diet, with or without tea, for 27 days. Iron absorption measured by whole-body counting before and after exposure showed that rats adapted to the high dietary level of FeSO4 or NaFeEDTA by down-regulating iron absorption to a similar extent. However, nonheme iron concentrations in liver and spleen were about 35-50% lower, whereas the concentration in kidney was about 300% higher in rats fed NaFeEDTA, compared to rats fed FeSO4. Tea had no major impact on iron absorption or iron status, regardless of iron source. Our results showed that although iron absorption was down-regulated similarly, body iron distribution was markedly different between rats exposed to FeSO4 and those exposed to NaFeEDTA. Further studies are warranted to determine the effects of prolonged exposure to dietary NaFeEDTA on kidney iron accumulation and kidney function.     

Combined impact of pH and organic acids on iron uptake by Caco-2 cells

Previous studies have shown that organic acids have an impact on both Fe(II) and Fe(III) uptake in Caco-2 cell. However, to what extent this effect is correlated with the anion of organic acids per se, or with the resulting decrease in pH, has not yet been clarified. Therefore, we studied the effect of five organic acids (tartaric, succinic, citric, oxalic, and propionic acid) on the absorption of Fe(II) and Fe(III) in Caco-2 cells and compared this with sample solutions without organic acids but set to equivalent pH by HCl. The results showed that the mechanisms behind the enhancing effect of organic acids differed for the two forms of iron. For ferric iron the organic acids promoted uptake both by chelation and by lowering the pH, whereas for ferrous iron the promoting effect was caused only by the lowered pH.     

National fortification of staple foods can make a significant contribution to micronutrient intake of South African adults

BACKGROUND: A national survey found that micronutrient deficiencies are prevalent in South African children, particularly calcium, iron, zinc, riboflavin, niacin, vitamin B6, folate, vitamin A, E and C. Mandatory fortification of maize meal and wheat flour were introduced in 2003 to combat some of the deficiencies found in children. To date however, there has not been a national survey on dietary intake in adults. OBJECTIVES: The main objectives of this study were to evaluate the micronutrient intake of the diet consumed by the average adult South African by means of secondary data analyses and secondly to evaluate the effects of fortification on selected nutrient intakes. STUDY DESIGN: Secondary data analysis was carried out with numerous dietary surveys on adults to create a database that included sampling (and weighting) according to ethnic/urban-rural residence in line with the population census, of which 79% were black Africans and the majority resided in rural areas. The effect of fortification was evaluated by substituting fortified foods in the diet for the unfortified products. SUBJECTS: The combined database used in this study comprised 3229 adults. RESULTS: Mean calcium, iron, folate and vitamin B6 intakes were very low particularly in women. Mean intakes of most micronutrients were lower in rural areas. Fortification of maize meal and wheat flour (bread) raised mean levels of thiamine, riboflavin, niacin, vitamin B6 and folate above the recommended nutrient intakes (RNIs). In women, despite fortification, mean iron intakes remained below the RNIs, as did calcium since it was not in the fortification mix. CONCLUSION: The average dietary intake of adults was of poor nutrient density, particularly in rural areas. Fortification of maize meal and wheat flour (bread) considerably improved mean vitamin B6, thiamine, riboflavin, niacin, folate and iron intakes as well as the overall mean adequacy ratio of the diet.     

Comparison of iron bioavailability from 15 rice genotypes: studies using an in vitro digestion/caco-2 cell culture model

An in vitro digestion/Caco-2 model was used to compare iron bioavailability from 15 selected Fe-dense and normal genotypes of unpolished rice from the International Rice Research Institute. Iron uptake was determined using Caco-2 cell ferritin formation in response to exposure to a digest of the cooked rice. Iron bioavailabilities from all rice genotypes were ranked as a percent relative to a control variety (Nishiki). Iron concentration in the rice samples ranged from 14 to 39 microg/g. No correlation was observed between Fe uptake and grain-Fe concentration. Furthermore, phytic acid levels were not correlated with Fe bioavailability. Genotypes with low Fe bioavailability (Tong Lan Mo Mi, Zuchein, Heibao, and Xua Bue Nuo) were noticeably more brown to purple in color. The results suggest that certain unknown compounds related to rice grain color may be a major factor limiting Fe bioavailability from unpolished rice.     

Interaction of Iron with Copper,Zinc, and Manganese in Wheat as Affected by Iron and Manganese in a Calcareous Soil

Iron (Fe) availability is low in calcareous soils of southern Iran. The chelate Fe-ethylenediamine di (o-hydroxy-phenylacetic acid) (Fe-EDDHA), has been used as an effective source of Fe in correcting Fe deficiency in such soils. In some cases, however, its application might cause nutritional disorder due to the antagonistic effect of Fe with other cationic micronutrients, in particular with manganese (Mn). A greenhouse experiment was conducted to evaluate the influence of soil and foliar applications of Fe and soil application of manganese (Mn) on dry matter yield (DMY) and the uptake of cationic micronutrients in wheat (Triticum aestivum L. var. Ghods) in a calcareous soil. Results showed that neither soil application of Fe-EDDHA nor foliar application of Fe sulfate had a significant effect on wheat DMY. In general, Fe application increased Fe uptake but decreased that of Mn, zinc (Zn), and copper (Cu). Application of Mn increased only Mn uptake and had no significant effect on the uptake of the other cationic micronutrients. Iron treatments considerably increased the ratio of Fe to Mn, Zn, Cu, and (Mn + Zn + Cu). Failure to observe an increase in wheat DMY following Fe application is attributed to the antagonistic effect of Fe with Mn, Zn, and Cu and hence, imbalance in Fe to (Mn + Zn + Cu) ratio. Due to the nutritional disorder and imbalance, it appears that neither soil application of Fe-EDDHA nor foliar application of Fe-sulfate is appropriate in correcting Fe deficiency in wheat grown on calcareous soils. Hence, growing Fe-efficient wheat cultivars should be considered as an appropriate practice for Fe chlorosis-prone calcareous soils of southern Iran.