Excess unabsorbed iron in the gastrointestinal tract may select for enteric pathogens and increase the incidence and severity of infectious disease. Aspergillus oryzae (Ao) is a filamentous fungus that has the ability to accumulate and store large amounts of iron, and when used as a supplement or fortificant, has similar absorption to ferrous sulphate (FeSO4) in humans. The objective of this study was to determine the effect of iron-enriched Ao (Ao iron) compared with FeSO4 on iron accumulation, growth and motility of the Gram-negative enteric pathogen, S. Typhimurium. S. Typhimurium was cultured in media containing no added iron or 1 μM elemental iron as either Ao iron or FeSO4. S. Typhimurium cultured with FeSO4 accumulated more iron than those cultured with Ao iron. Genes regulated by the iron-activated transcriptional repressor, Fur, did not differ between control and Ao iron, but decreased in S. Typhimurium cultured with FeSO4 compared with both groups. Growth of S. Typhimurium was greater when cultured with FeSO4 compared with Ao iron and control. S. Typhimurium swam faster, had greater acceleration and travelled further when cultured with FeSO4 compared with Ao iron and control; swim speed, acceleration and distance travelled did not differ between Ao iron and control. These findings provide evidence that Ao iron reduces the virulence of a common enteric pathogen in vitro. Further research is required to determine whether iron-enriched Ao is a suitable iron supplement to improve iron delivery in areas with a high infection burden.

Fe fortification of centrally manufactured and frequently consumed condiments such as bouillon cubes could help prevent Fe deficiency in developing countries. However, Fe compounds that do not cause sensory changes in the fortified product, such as ferric pyrophosphate (FePP), exhibit low absorption in humans. Tetra sodium pyrophosphate (NaPP) can form soluble complexes with Fe, which could increase Fe bioavailability. Therefore, the aim of this study was to investigate Fe bioavailability from bouillon cubes fortified with either FePP only, FePP+NaPP, ferrous sulphate (FeSO4) only, or FeSO4+NaPP. We first conducted in vitro studies using a protocol of simulated digestion to assess the dialysable and ionic Fe, and the cellular ferritin response in a Caco-2 cell model. Second, Fe absorption from bouillon prepared from intrinsically labelled cubes (2·5 mg stable Fe isotopes/cube) was assessed in twenty-four Fe-deficient women, by measuring Fe incorporation into erythrocytes 2 weeks after consumption. Fe bioavailability in humans increased by 46 % (P<0·005) when comparing bouillons fortified with FePP only (4·4 %) and bouillons fortified with FePP+NaPP (6·4 %). Fe absorption from bouillons fortified with FeSO4 only and with FeSO4+NaPP was 33·8 and 27·8 %, respectively (NS). The outcome from the human study is in agreement with the dialysable Fe from the in vitro experiments. Our findings suggest that the addition of NaPP could be a promising strategy to increase Fe absorption from FePP-fortified bouillon cubes, and if confirmed by further research, for other fortified foods with complex food matrices as well.

In addition to phytate, polyphenols (PP) might contribute to low Fe bioavailability from sorghum-based foods. To investigate the inhibitory effects of sorghum PP on Fe absorption and the potential enhancing effects of ascorbic acid (AA), NaFeEDTA and the PP oxidase enzyme laccase, we carried out three Fe absorption studies in fifty young women consuming dephytinised Fe-fortified test meals based on white and brown sorghum varieties with different PP concentrations. Fe absorption was measured as the incorporation of stable Fe isotopes into erythrocytes. In study 1, Fe absorption from meals with 17 mg PP (8·5 %) was higher than that from meals with 73 mg PP (3·2 %) and 167 mg PP (2·7 %; P< 0·001). Fe absorption from meals containing 73 and 167 mg PP did not differ (P= 0·9). In study 2, Fe absorption from NaFeEDTA-fortified meals (167 mg PP) was higher than that from the same meals fortified with FeSO4 (4·6 v. 2·7 %; P< 0·001), but still it was lower than that from FeSO4-fortified meals with 17 mg PP (10·7 %; P< 0·001). In study 3, laccase treatment decreased the levels of PP from 167 to 42 mg, but it did not improve absorption compared with that from meals with 167 mg PP (4·8 v. 4·6 %; P= 0·4), whereas adding AA increased absorption to 13·6 % (P< 0·001). These findings suggest that PP from brown sorghum contribute to low Fe bioavailability from sorghum foods and that AA and, to a lesser extent, NaFeEDTA, but not laccase, have the potential to overcome the inhibitory effect of PP and improve Fe absorption from sorghum foods.

Adverse sensory changes prevent the addition of highly bioavailable ferrous sulfate (FeSO4) to most wheat flours. Poorly absorbable reduced Fe powders are commonly used. Encapsulation of FeSO4 can overcome these sensory changes, but the particle size of commercial compounds is too large to be used by flour mills. The first objective of the study was to measure the efficacy in wheat flour of two newly developed Fe compounds, an H-reduced Fe powder (NutraFine™ RS; North America Höganäs High Alloys LLC, Johnstown, PA, USA) and small particle-sized (40 μm) encapsulated FeSO4. As a second objective, the microcapsules were evaluated as a vehicle for iodine fortification. A randomised, double-blind controlled intervention trial was conducted in Kuwaiti women (n 279; aged 18–35 years) with low body Fe stores (serum ferritin (SF) < 25 μg/l) randomly assigned to one of three groups (20 mg Fe as NutraFine™ RS, 10 mg Fe as encapsulated FeSO4 and 150 μg iodine, or no fortification Fe) who consumed wheat-based biscuits 5 d per week. At baseline and 22 weeks, Hb, SF, transferrin receptor, urinary iodine and body Fe stores were measured. Relative to control, mean SF in the encapsulated FeSO4 group increased by 88 % (P < 0·001) and body Fe stores increased from − 0·96 to 2·24 mg/kg body weight (P < 0·001), while NutraFine™ RS did not significantly increase SF or body Fe stores. The median urinary iodine concentration increased from 140 to 213 μg/l (P < 0·01). NutraFine™ RS added at double the amount of Fe as FeSO4 was not efficacious in improving Fe status. The newly developed microcapsules were highly efficacious in improving both Fe stores and iodine status.

Previous data from Vietnam show that anaemia is highly prevalent among schoolchildren, who are considered not to be iron deficient. Trichuris infection doubled the risk of anaemia. The present study aimed to evaluate the hypothesis that de-worming is more effective than iron fortification in an anaemic, infection-prone population. In a trial with a 2 × 2 factorial design, 425 anaemic children aged 6–8 years were randomly assigned to receive either iron-fortified noodles or placebo, and mebendazole or placebo. Outcomes considered were change in haematological indicators of iron status (Hb, serum ferritin (SF), serum transferrin receptor (TfR) and haemoglobinopathies analysis); inflammations (C-reactive protein (CRP)); parasite infection status (hookworm, Trichuris and Ascaris infection); and IgE. ANOVA and logistic regression were used to assess the effects of iron fortification and de-worming on Hb, SF, TfR, body iron and anaemia. Hb improved in all groups after 6 months of intervention. Iron fortification significantly improved Hb, SF and body iron (2·6 g/l, 16·3 μg/l and 1 mg/kg, respectively). Prevalence of elevated IgE was very high at baseline (99 %) and significantly reduced to about 75 % in all groups after intervention. De-worming unexpectedly showed no effect on Hb, iron status and IgE level. It is concluded that iron fortification slightly improved anaemia and iron status in anaemic schoolchildren in rural Vietnam that were not considered iron deficient. Chronic infection or other unidentified factors may play an important role in the seasonal reduction of anaemia seen in all treatment groups.

Food fortification is an important long-term strategy for addressing micronutrient deficiencies. Finding the ideal Fe fortification compound, however, remains a challenge. In the present study the effect of ferrous bisglycinate as fortificant in brown bread was compared with that of electrolytic Fe among Fe-deficient school children in a randomised controlled trial. Children (n 160), aged 6–11 years, with serum ferritin <20μg/l, were randomly assigned to one of three treatment categories: (i) standard unfortified bread; (ii) bread with electrolytic Fe as fortificant; and (iii) bread with ferrous bisglycinate as fortificant. Each child received four slices of bread (120g) on school days, which supplied an average of 3·66mg elemental Fe per intervention day for 137d (2·52mg/d for 75d and 5·04mg/d for 62d) over a period of 7·5 months. Hb, serum ferritin, serum Fe and transferrin saturation were measured at baseline and at the end of the intervention. Significant treatment effects were observed for Hb (P=0·013), serum Fe (P=0·041) and transferrin saturation (P=0·042) in the ferrous bisglycinate group, but not in the electrolytic Fe group. There were no significant intervention effects for serum ferritin in either treatment group. Overall, ferrous bisglycinate as Fe fortificant in brown bread performed better than electrolytic Fe in a group of Fe-deficient school children over a period of 7·5 months.

Fe absorption is affected by many dietary factors. The objective of the present study was to measure the effects of high v. low content of vitamin C, meat and phytic acid in whole diets with Fe-fortified bread on the efficacy of Fe absorption. Thirty-two healthy women with low Fe stores were randomised to three groups, each of which was given two of six test diets containing either low/high amounts of vitamin C, meat or phytic acid, respectively, in a cross-over design. Each diet was served throughout a 5 d period. Fe-fortified rye bread, extrinsically labelled with 59Fe, was given with all main meals. Fe absorption was determined from whole-body counter measurements of 59Fe retention. The fractional non-haem Fe absorption (corrected to a 40% standard absorption by measurements from the reference dose) was 1·9% v. 3·4% (P=0·04) for the low/high vitamin C diets, 3·0% v. 3·5% (P=0·58) on the low/high meat diets and 4·9% v. 3·8% (P=0·24) on the low/high phytic acid diet, respectively. The total Fe absorbed (geometric mean with standard error) varied from 0·43 (se 0·11) mg from the diet with lowest bioavailability to 1·09 (se 0·18) mg from the diet with highest bioavailability (P<0·001). The present whole-diet study indicates that diet composition is a strong predictor of Fe absorption. In the diet with a low content of enhancers and a high content of inhibitors, vitamin C improved non-haem Fe absorption. The total Fe absorption varied 2·5-fold after small alterations of the content of enhancers and inhibitors in the diet.

In this three-phase study we first compared the availability of fourteen Fe forms in a wheat-based ready-to-eat breakfast cereal using an in vitro digestion/human colonic adenocarcinoma (CaCo-2) cell model. We then investigated the effect of milk and/or coffee on those fortified cereals found in phase 1 to show promising increases in Fe availability. The Fe forms assessed in phase 1 were reduced (control), carbonyl, electrolytic, FePO4, FeSO4, FeCO3, Na2FeEDTA, Ferrochel® (Albion Laboratories, Clearfield, UT, USA; ferrous bis-glycinate), encapsulated ferrous fumarate, FeSO4, ferrous lactate and Biofer® (LipoTech, Britwell Salome, Oxfordshire; FeSO4), SQM® (Sea-Questra-Min Iron; Quali Tech, Chaska, MN, USA; polysaccharide-complexed FeSO4) and Sun Active® (Taiyo Kagaku, Yokkaichi, Japan). All these forms increased Fe uptake compared with the unfortified cereal. Relative to the control, the following increases in Fe availability were observed: electrolytic, 52 %; ferrous fumarate, 30–35 %; Sun Active, 78 %; Ferrochel, 125 %; Na2FeEDTA, 291 %. Recent human studies have shown similar data with regard to Ferrochel, FeSO4 and Na2FeEDTA, with the latter being more bioavailable. Our phase-2 studies indicated that the addition of milk to FeSO4-fortified cereal increased Fe availability, but this availability was markedly decreased by the addition of coffee to the digest. Conversely, a loss in availability from Na2FeEDTA was observed with the addition of milk; however, the addition of coffee did not markedly affect Fe availability from this form. In phase-3 studies we observed increased Fe availability upon the addition of milk to cereals containing Ferrochel, FeSO4, Sun Active, a mixture of reduced Fe and Na2FeEDTA or reduced Fe. For these forms we did not assess the behaviour after the addition of coffee. In conclusion, when considering possible fortificants for optimizing Fe bioavailability within a foodstuff, it is of paramount importance to consider the interaction between the fortified foodstuffs and other components of the meal (such as milk and coffee with a breakfast).

Ferric pyrophosphate is a water-insoluble Fe compound used to fortify infant cereals and chocolate-drink powders as it causes no organoleptic changes to the food vehicle. However, it is only of low absorption in man. Recently, an innovative ferric pyrophosphate has been developed (Sunactive Fe™) based on small-particle-size ferric pyrophosphate (average size 0·3 μm) mixed with emulsifiers, so that it remains in suspension in liquid products. The aim of the present studies was to compare Fe absorption of micronised, dispersible ferric pyrophosphate (Sunactive Fe™) with that of ferrous sulfate in an infant cereal and a yoghurt drink. Two separate Fe absorption studies were made in adult women (ten women/study). Fe absorption was based on the erythrocyte incorporation of stable isotopes (57Fe and 58Fe) 14 d after the intake of labelled test meals of infant cereal (study 1) or yoghurt drink (study 2). Each test meal was fortified with 5 mg Fe as ferrous sulfate or micronised, dispersible ferric pyrophosphate. Results are presented as geometric means. There was no statistically significant difference between Fe absorption from micronised, dispersible ferric pyrophosphate- and ferrous sulfate-fortified infant cereal (3·4 and 4·1 % respectively; P=0·24) and yoghurt drink (3·9 and 4·2 % respectively; P=0·72). The results of the present studies show that micronised, dispersible ferric pyrophosphate is as well absorbed as ferrous sulfate in adults. The high relative Fe bioavailability of micronised, dispersible ferric pyrophosphate indicates the potential usefulness of this compound for food fortification.

Ascorbic acid and Na2EDTA enhance Fe absorption from the water-soluble Fe compound FeSO4 but their effect on poorly water-soluble Fe compounds such as ferrous fumarate is less well established. In the present study, the effects of ascorbic acid and Na2EDTA on Fe absorption from ferrous fumarate were evaluated in adult women (ten women/study) from the erythrocyte incorporation of Fe stable isotopes (57Fe or 58Fe) 14 d after administration. Two separate studies were made with test meals of Fe-fortified infant cereal (5 mg Fe/meal). Data were evaluated by paired t tests and the results are presented as geometric means. In study 1a, the comparison between Fe absorption from ferrous fumarate- and FeSO4-fortified cereal showed that adult women absorb Fe as well from ferrous fumarate as from FeSO4 (3·0 and 3·1 % respectively, P=0·85). After addition of Na2EDTA (Na2EDTA:fortification Fe molar ratio of 1:1), Fe absorption from FeSO4 was significantly higher than from ferrous fumarate (5·3 v. 3·3 % respectively, P<0·01; study 1b). In study 2, Fe absorption was compared from ferrous fumarate-fortified meals with and without ascorbic acid added at a 4:1 molar ratio (relative to fortification Fe) and the results showed that ascorbic acid increased Fe absorption from ferrous fumarate significantly (6·3 v. 10·4 %, P=0·02). The results of the present studies show that Fe absorption from ferrous fumarate is enhanced by ascorbic acid but not by Na2EDTA, thus emphasising that not all findings from Fe absorption studies made with FeSO4 can be extrapolated to Fe compounds with different solubility properties.

Fe absorption was measured in adult human subjects consuming different cereal foods fortified with radiolabelled FeSO4, ferrous fumarate or NaFeEDTA, or with radiolabelled FeSO4 or ferric pyrophosphate in combination with different concentrations of Na2EDTA. Mean Fe absorption from wheat, wheat–soyabean and quinoa (Chenopodium quinoa) infant cereals fortified with FeSO4 or ferrous fumarate ranged from 0·6 to 2·2 %. For each infant cereal, mean Fe absorption from ferrous fumarate was similar to that from FeSO4 (absorption ratio 0·91–1·28). Mean Fe absorption from FeSO4-fortified bread rolls was 1·0 % when made from high-extraction wheat flour and 5·7 % when made from low-extraction wheat flour. Fe absorption from infant cereals and bread rolls fortified with NaFeEDTA was 1·9–3·9 times greater than when the same product was fortified with FeSO4. Both high phytate content and consumption of tea decreased Fe absorption from the NaFeEDTA-fortified rolls. When Na2EDTA up to a 1:1 molar ratio (EDTA:Fe) was added to FeSO4-fortified wheat cereal and wheat–soyabean cereal mean Fe absorption from the wheat cereal increased from 1·0 % to a maximum of 5·7 % at a molar ratio of 0·67:1, and from the wheat–soyabean cereal from 0·7 % to a maximum of 2·9 % at a molar ratio of 1:1. Adding Na2EDTA to ferric pyrophosphate-fortified wheat cereal did not significantly increase absorption (P>0·05). We conclude that Fe absorption is higher from cereal foods fortified with NaFeEDTA than when fortified with FeSO4 or ferrous fumarate, and that Na2EDTA can be added to cereal foods to enhance absorption of soluble Fe-fortification compounds such as FeSO4.

An evaluation was made into the usefulness of ferrous fumarate as an iron fortificant for an experimental chocolate drink powder targetted to children and adolescents. Organoleptically ferrous fumarate was acceptable when the chocolate drink powder was reconstituted in milk or water that was heated to < 80°. Unacceptable colour changes occurred, however, when boiling milk or water were used. In human Fe absorption studies when the Fe compounds were added to the chocolate drink immediately before consumption, ferrous fumarate was 3.31 % absorbed compared with 2.82% for ferrous sulphate and 2.11 % for ferric pyrophosphate. When the Fe compounds were processed during the manufacture of the chocolate drink powder, the absorption of ferrous fumarate was 5.27 %, ferrous sulphate 2.62 % and ferric pyrophosphate 0.55 %. Ascorbic acid had little or no effect on the absorption of ferrous fumarate. It is concluded that food processing can influence the relative absorption of fortification Fe and that, if not reconstituted with boiling milk or water, ferrous fumarate could be a useful compound for the fortification of chocolate drink powders.