Challenges and strategies to combat global iron deficiency by food fortification

Iron is an important micronutrient required for healthy living. Therefore, populations dependent primarily on plant-based diets with little or no animal flesh may become deficient in iron. It is found that ~ 30% of the world population suffers from iron deficiency anemia. Since iron undergoes oxidation which reduces its absorption, formulation of iron supplements and fortificants for treatment of iron deficiency has been studied extensively. Iron fortified food has been introduced in order to overcome the iron deficiency. Chelation of iron with amino acids, EDTA, fumarate, succinate, and gluconate and encapsulating iron by inclusion complexation and liposome encapsulations have improved the bioavailability of iron in the human body. Among these methods, iron delivery via complexation and encapsulation are common due to their high efficacy


INTRODUCTION
Natural food consumption has undergone a metamorphosis from fresh plucked, home grown to highly preserved and long shelf-life products.Thus, nutrient loss is a concern resulting in food supplementation and fortification.Food supplements in the forms of pills, tablets, capsules, or as liquids in syrup or suspensions are taken in addition to those obtained in the normal diet as concentrated nutrients for nutritional or physiological effect.A fortificant on the other hand, is a nutrient added over the natural level of the nutrient present in the food for the purpose of increasing its nutrient content.Thus, cereals are fortified with iron and vitamins.Iron is used as a food supplement as well as a fortificant in various food items.Use as a fortificant requires consideration of many factors.The primary factor being sensory problems where unacceptable colour, texture, flavor, or odor may affect the final product.Iron has the likelihood of interactions with components in the food matrix which can affect its stability and make it segregate out of the food matrix (Table 1).In this context, the delivery of iron in the form of supplements and fortificants need careful preparation and processing.Here, we review the different forms of iron delivery methods available along with new developments in delivery techniques.

IMPORTANCE OF IRON AS A NUTRIENT
Iron has the capability of existing at various oxidation states from -2 to +6.However, in biological systems, iron exists mainly in two oxidation states, ferrous state (+2) and ferric state (+3) (Beard, 2001).It is a functional component in many biologically important macromolecules such as hemoglobin, myoglobin, cytochromes, oxygenases, flavoproteins, and redoxins (Vashchenko and MacGillivray, 2013) and is important for cell growth and differentiation.As iron can participate in many electron transfer reactions in the body, it plays a central role in biological processes such as respiration, DNA synthesis, and energy production.Iron can facilitate redox reactions by changing its oxidation state from reduced ferrous state to oxidized ferric state or vice versa (Mackenzie et al., 2008).In addition, this ability to oscillate from one oxidation state to the other allows iron to reversibly bind ligands such as oxygen, nitrogen, and sulphur atoms (Beard, 2001).

REVIEW ARTICLE
Table 1: Types of iron fortified foods and their iron contents.

Fortified food Iron source/s Added iron content Reference
Curry powder NaFeEDTA 25 µg Fe/g curry powder (Ballot et al., 1989) Cheddar cheese Ferric chloride 90 mg Fe/g cheddar cheese (Zhang and Mahoney, 1989) Fe-casein complex 29 mg Fe/g cheddar cheese Ferripolyphosphate-whey protein complex 36 mg Fe/g cheddar cheese

Salt
Ferrous sulfate 1 mg Fe/g salt (Rao, 1994) Bread rolls FeSO 4 and NaFeEDTA 2.5 mg Fe/50 g wheat flour (Hurrell et al., 2000) Infant cereal Ferrous succinate 50 mg Fe/100 g rice cereal (Hurrell et al., 1989 Iron containing proteins in the mammalian system carry out functions that are essential for the maintenance of the body.Since iron is an essential constituent in cytochromes that participate in ATP production, it is necessary for efficient energy production in the body.Iron present as a cofactor in heme, is the largest iron reservoir in the body (Johnson and Wessling-Resnick, 2012).Haemoglobin that contains iron is important to transport oxygen throughout the body.The porphyrin prosthetic groups that contain iron as the metal center, reversibly bind the dioxygen ligand and carry the oxygen from the lungs to the tissues.Myoglobin is required for oxygen storage in the body and for the use of oxygen in the muscles (Alaunyte et al., 2014).Iron is a central component in the heme enzymes.Cytochromes A, B, and C contain heme as the active site and carry out electron transfer employing the variable oxidation states of iron.Additionally, iron facilitates transfer of electrons to molecular oxygen at the end of the respiratory chain (Geissler and Singh, 2011).The catalases and peroxidases which facilitate breakdown of hydrogen peroxide also contain iron in them.
Most single electron transfer reactions are facilitated by iron-sulphur complexes present in the enzymes.Aconitase, an enzyme required for the function of the citric acid cycle, ribonucleotide reductase essential for DNA synthesis, phenylalanine hydroxylase, and tyrosine hydroxylase required for melanin synthesis are some examples of biologically important iron-sulphur complexes (Geissler and Singh, 2011).Iron is also found to be an important contributor in immune responses (Mackenzie et al., 2008).

Consequences of iron deficiency
According to World Health Organization (WHO), iron deficiency anemia is the most common micronutrient deficiency throughout the world.Over two billion people in the world have been diagnosed with anemia.
In Sri Lanka, a survey performed in 2009 has shown that 25.2% of children under the age of five, suffer from iron deficiency anemia (Jayatissa et al., 2012).The daily requirement of iron intake depends on the gender and age and the estimated average iron requirement is given in Table 2. Iron deficiency anemia impairs the activity of many enzymes, decreases myoglobin in skeletal muscle, and lowers the level of hemoglobin in the bloodstream (Beard, 2001), resulting in decreased physical activity, lethargy and increased risk of infection due to impaired immune response.Anemia also affects the early physical and cognitive development of infants (Beinner and Lamounier, 2003).The morbidity and mortality of pregnant women at the time of childbirth is shown to increase due to iron deficiency (Hurrell et al., 2000), and therefore, responsible for about 26% of the maternal deaths around the world (Brabin et al., 2001).Low iron content in blood is also linked to inflammatory bowel diseases, and one third of these patients have been found to suffer from anemia (Gasche, 2004).Further, iron deficiency can cause chronic kidney diseases (Babitt and Lin, 2012) and heart failure (Jankowska et al., 2013).Various measures taken to minimize iron deficiency include intake of iron supplements, fortification of food with iron, and biofortification of crops (Allen, 2002).

Dietary sources and iron metabolism
There are two forms of dietary iron, namely heme iron and non-heme iron.It is found that the relative absorptivity is higher in heme iron than non-heme iron in the human body (Hurrell and Egli, 2010).This distinction is made according to the mechanism of absorption of iron in the body.A certain percentage of iron present in meat, fish, and poultry is heme iron.Plant-based foods such as fruits and vegetables contain only non-heme iron, also known as inorganic iron, in the form of iron salts and chelates (Lim et al., 2013).
Inorganic iron forms about 90% of the total iron absorbed from food (Tandara and Salamunic, 2012).Heme iron is absorbed as a porphyrin complex into the mucosal cells in the small intestine (Lim et al., 2013).Non-heme iron in ferrous state is transported using divalent metal transporter 1 (DMT1), and non-heme ferric iron is converted to ferrous form by duodenal cytochrome reductase (Geissler and Singh, 2011).Then, the ferrous ion enters an exchangeable iron pool in the enterocyte, from where iron will be supplied as required for various functions.A regulated mechanism for excretion of iron is unknown to date and therefore the content of iron in the body is controlled by the absorption at the small intestine (Tandara and Salamunic, 2012).Heme iron remains in the porphyrin complex until it is absorbed by the mucosal cells; the influence of external factors on heme iron absorption is minimal.However, non-heme iron absorption is influenced by factors that enhance or inhibit iron solubility (Osungbade and Oladunjoye, 2012).

Factors affecting iron absorption
Absorption of iron in the intestines can be affected by the presence of several dietary factors associated with the food matrix.Since heme iron is associated with the prophyrin ring, absorption of heme iron is greater than that of non heme iron.Ascorbic acid binds iron and keeps it in the reduced ferrous state and thereby enhances its bioavailability.Similarly compounds such as glycine, citrate, gluconate, and fumarate which can chelate ferrous iron and improve its solubility may increase iron bioavailability.On the other hand, plant components such as polyphenols, phosphates, calcium, and dietary fiber are known to reduce non-heme iron absorption into the body (Chen and Oldewage-Theron, 2002;Hurrell et al., 1999;Roughead et al., 2002) through the formation of insoluble iron salts/complexes.

Ascorbic acid
Ascorbic acid has been found to drastically increase iron absorption from meals.According to findings from the research carried out by Hallberg et al. in 1987, there is a semiexponential relationship between the amount of ascorbic acid in the meal and the amount of iron absorbed.Another study was carried out by Derman et al. (1977) on 116 Indian women to determine the iron absorption from maize meal porridge containing ascorbic acid .The meals were given with and without tea or coffee and the iron absorption was measured using a radio-iron utilization method.The research revealed that addition of 50-100 mg of ascorbic acid would increase iron absorption by ten-fold when taken without tea, and that using doses of 250-500 mg of ascorbic acid could even overcome the inhibitory effect of tea on iron absorption.Two mechanisms have been suggested to be responsible for the increase in iron absorption in the presence of ascorbic acid.The first mechanism is the formation of soluble ironascorbic acid complexes.Absorption of nonheme iron depends on its extent of solubility within the lumen of the upper intestinal tract.Thus, the formation of soluble ascorbic acid iron complexes will lead to an increase in iron absorption.The second mechanism by which ascorbic acid increases iron absorption is reducing iron in ferric to ferrous form.Also, ascorbic acid prevents binding of other ligands in the intestine with iron, which may reduce iron absorption.However, ascorbic acid is unstable under the temperature and humidity conditions used in food processing.Therefore, the use of ascorbic acid in processed food to increase iron absorption may not be fruitful.A study carried out by Lynch and Cook demonstrated that ascorbic acid is effective only if it is present in the meal.They observed a six-fold increase in iron absorption when 500 mg of ascorbic acid is taken with the meal.However, when the same amount of ascorbic acid was taken 4 to 8 hours prior to the meal, its effect on iron absorption was found to be insignificant (Lynch and Cook, 1980).Human studies using radioisotopes carried out by Lynch and Stoltzfus (2003) concluded that the increase of iron absorption by ascorbic acid depends on the dose of ascorbic acid consumed and that during iron absorption, ascorbic acid needs to be present in the lumen of the upper gastrointestinal tract.They also found that intravenously injected ascorbic acid does not demonstrate an increase in iron absorption.Meat is also known to increase absorption of nonheme iron to the body.

Phytates
Phytates are powerful chelators which can bind on to metals and form insoluble complexes.Due to its formation of insoluble complexes with iron cations, phytate is considered to be an inhibitor of iron absorption.Cereals and legumes are known to contain phytates and phytic acid.Koreissi-Dembele et al. (2013) studied the effect of dephytinization on iron absorption in fonio meals.Incubation at pH 5, 50 o C was found to degrade phytic acid.Iron absorption was shown to increase from 2.6% in the non-dephytinized meal to 8.3% in the dephytinized meal (Koreissi-Dembele et al., 2013).

IRON DELIVERY METHODS
When dietary intake of iron is insufficient to meet the bodily requirements, iron is taken as a supplement and also iron fortification is carried out on staple food (Beinner and Lamounier, 2003;Mirmiran et al., 2012).Oral iron supplements of widely varying dosages, formulations (quick or prolonged release), and chemical states (ferrous or ferric form) are available (Table 3).A study on the bioavailability of the ferrous and ferric forms has shown that slow-release ferrous sulphate preparations have good bioavailability, efficacy, and acceptable tolerability when compared with ferric iron polymaltose complex preparations (Santiago, 2012).
However, iron can change color of food by forming complexes with compounds such as sulphur compounds, tannins, and polyphenols.Iron is chemically reactive under alkaline conditions, give a metallic taste, cause gastrointestinal discomfort, and give an unpleasant flavor due to fat oxidation (Chen and Oldewage-Theron, 2002;Mehansho, 2006).Also, iron in ferrous state may be easily oxidized into less bioavailable ferric state.Moreover, interaction of non-heme iron with various dietary components in the intestine may reduce the solubility and therefore, lower the bioavailability.To minimize these drawbacks, chelation of iron with biocompatible ligands and encapsulation of iron with various edible coatings have been carried out (Mehansho, 2006).We present some of these methods which have shown promise as effective carriers or delivery agents of ferrous iron.

Chelation
Iron chelation is a technique used to deliver active ingredients that have either low absorption efficiency or are less bioavailable.Chelation is literally the protective enclosure of minerals such as iron and zinc, so that these minerals do not undergo any unnecessary reactions before reaching the target.A set of chelating agents such as EDTA (Ethylenediaminetetraacetate), chitosan, amino acids, gluconate, succinate, fumarate are used in chelating cations that need to be delivered (Flora, 2013).Ferrous sulphate is very soluble and therefore has high bioavailability.However, it is also susceptible to organoleptic changes and interactions with ligands in the intestine to form insoluble complexes.To minimize these unfavorable reactions, chelation of iron is carried out.

Amino acid chelated iron
Use of amino acids to chelate iron causes iron to be absorbed in the jejunum, unlike the non-heme iron which is absorbed in the duodenum.One example of an amino acid chelated iron complex is ferrous bis-glycinate, in which one ferrous cation is chelated with two glycine ligands.Glycine too is capable of protecting iron from binding on to ligands that can reduce iron absorption.Benjamin et al. (2000)carried out a study using 10 iron sufficient males to compare the efficiencies of iron absorption from ferrous sulphate, ferrous bis-glycinate, and ferric trisglycinate from a phytate-rich meal.The subjects were fed whole maize porridge fortified with 59 Fe-sulphate on day one and 59 Fe bis-glycinate on the second day.Then iron absorption was determined from blood radioactivity.The results showed that iron absorption from ferrous bisglycinate is four times that of ferrous sulphate.Ferrous bis-glycinate is water soluble, and therefore can easily oxidize from ferrous to ferric state resulting in color and flavor changes (Jiménez-Alvarado et al., 2009;Mehansho, 2006).

EDTA complexes
EDTA ligand has been found to form stable complexes with iron.EDTA is a hexadentate chelate ligand and is capable of forming complexes with many metal cations (Chen and Oldewage-Theron, 2002).Findings from a study carried out by Hurrell et al. (2000) demonstrated that the complex NaFeEDTA facilitated higher iron absorption than that from ferrous sulphate and ferrous fumarate.Eighty-four volunteers from ages between 18-40 years were given infant cereals and bread rolls fortified with radio labeled ferrous sulphate or ferrous fumarate and Fe absorption was measured based on erythrocyte enrichment.Then the subjects were fed infant cereals and bread rolls fortified with radio labeled NaFeEDTA.Iron absorption from NaFeEDTA fortified meal was observed to be 1.9-3.9times greater than that from ferrous sulphate or ferrous fumarate fortified meal.It was also demonstrated that addition of Na 2 EDTA can increase iron absorption from cereal fortified with ferrous sulphate.Iron-EDTA complexes are stable and the bioavailability of iron from the complex is not affected by the harsh conditions used in food processing.They are advantageous since they cause fewer organoleptic problems.Presence of EDTA ligand can minimize the effect of phytates on iron absorption.EDTA ligand is not metabolized in the body, and is excreted through the urinary tract.Divalent cations bind EDTA with high affinity and therefore, loss of biologically important divalent cations may occur.This process is named co-chelation (Chen and Oldewage-Theron, 2002).Since, iron-EDTA complexes are about six times more expensive than ferrous sulphate (Le et al., 2006), the use of EDTA as a ligand has its restrictions.

Fumarate, succinate, and gluconate ligands
Unless chelated or protected with a coating, iron in ferrous state is easily oxidized into ferric state.Ferric iron can precipitate proteins and cause gastro intestinal irritation when ingested.However, the precipitation of proteins does not occur when iron is chelated with gluconate or succinate ligands.Therefore, succinate and gluconate complexes of iron are used to deliver iron (Hurrell et al., 1989;Khosroyar and Arastehnodeh, 2007).Ferrous fumarate is commonly used in iron supplements due to its high bioavailability (Chen and Oldewage-Theron, 2002).Also, since fumarate ligand is present in living organisms and is an essential component in Krebs's cycle, ferrous fumarate shows good biocompatibility and minimal side effects (Kapor et al., 2012).

Encapsulation
Encapsulation involves creating a protective layer around a particular substance.In food and pharmaceutical applications this protective coating can assist controlled release of the entrapped substance of interest.So far, various encapsulation techniques such as polymerization, spray drying, gelation, precipitation, and coacervation with relation to food industry have been reported (Ariyarathna and Karunaratne, 2015;Bastos et al., 2012;Karunaratne et al., 2016;Gadkari and Balaraman, 2015).Encapsulated iron compounds have been used in iron fortification to minimize interactions between the other components in the food vehicle (Chen and Oldewage-Theron, 2002).When choosing the shell material for the capsule, it is important to consider its solubility in gastric juice.Whey protein, chickpea protein, alginate, and hydrogenated oils have been used to encapsulate iron containing compounds (Bezbaruah et al., 2011;Khosroyar and Arastehnodeh, 2007;Martin and Jong, 2012;Mehansho, 2006;Rajakaruna et al., 2015;Zimmermann et al., 2004).Various techniques of encapsulation have been utilized in studies to increase the bioavailability and to solve organoleptic issues related to iron.
Cold-set gelation has been used to entrap iron in the presence of ascorbic acid which can otherwise undergo degradation on heat application.Whey protein has been used to create gel particles by first unfolding the protein structure and then inducing cold-set gelation by addition of Fe 2+ and ascorbic acid.Particles have then been freeze dried at -40 o C. Presence of ascorbate has been found to increase the strength of the iron induced cold-set gel.The particles have also displayed protein stability at pH range 2 to 7, indicating high encapsulation efficiency.Also, the presence of ascorbic acid in gel particles has resulted in an increase in in vitro iron accessibility from 10 to 80% (Martin and Jong, 2012).
Alginate has been used to encapsulate iron.The carboxylic groups on the alginate molecules and ferric cations can crosslink, creating a capsule with the iron compound at its core, and alginate as the shell.Alginate is insoluble in aqueous media.Therefore, the shell can protect the inner iron compound from interacting with moisture, and reduce organoleptic changes iron may cause.Khosroyar et al. (2007) have used coacervation to form ferric saccharate microcapsules using an alginate coating.Here, alginate and ferric saccharate have been mixed at a shell: core ratio of 70:30, and added drop wise to a solution of CaCl 2 .The particles formed have then been washed to remove free ions and dried.The alginate coated ferric saccharate particles have shown an average size of 400 µm.Iron release from the capsules under wet or dry conditions at room temperature has been found to be less than 0.04%.When the amount of water in contact with the capsules is increased, a higher release has been observed.Zimmerman et al. (2004) have reported encapsulating iron along with iodine and vitamin A, to be used as a food fortificant.They used hydrogenated palm oil to coat iron and other components employing a single step spray cooling technique.The resultant capsules had a mean particle size of 100 µm and were spherical in shape.On storage for six months, the particles only lost 12-15% of its iron content.
Encapsulation has also been attempted using emulsion formation.Jimenez-Alvarado et al. (2009) have reported entrapping ferrous bisglycinate solution in the inner aqueous phase of water-in-oil-in-water (w/o/w) emulsions .As discussed previously, despite being able to reduce interactions of iron with phytates and polyphenols, ferrous bis-glycinate can cause color changes in food, and is also oxidized easily.Water-in-oil-in-water emulsions are made from dispersing water droplets in large oil droplets, and then dispersing the oil droplets in an aqueous phase.When biopolymers such as proteins are used in the oil phase, chemical interactions through covalent bonding, or physical interactions through electrostatic forces can take place (Dickinson, 2008).Therefore, protein-hydrocolloid systems are increasingly being used in food processing.Chickpea protein coated ferrous fumarate microparticles with high encapsulation efficiency and good release kinetics at pH 2, 4, 6, and 8 have been synthesized by Rajakaruna et al. (2015).The isoelectric precipitation technique of adjusting the pH of a homogeneous protein and ferrous fumarate solution to the pI of the protein has been employed to obtain the encapsulate (Rajakaruna et al., 2015).β-Cyclodextrin is a cyclic carbohydrate which has a conical cave-like conformation as depicted in figure 4(b).This shape enables β-cyclodextrin to accommodate active ingredients in its central cavity.β-Cyclodextrins contain hydrophilic moieties directed outwards and a lipophilic interior which provides a suitable environment to solubilize organic compounds.The entrapped hydrophobic molecules can easily dissolve in the aqueous media, thereby increasing the stability, solubility, and bioavailability of the hydrophobic molecule.This technique is mainly used to encapsulate organic compounds such as vitamins, essential oils, and some poor water soluble drugs.Compared to other methods, loading efficiency is typically low, but the encapsulation efficiency and the stability of the encapsulated compound against heat is relatively high (Polyakov and Kispert, 2015).Kapor et al. (2012) reported formation of ferrous fumarate inclusion complexes using cyclodextrins.To form the inclusion complexes, coprecipitation method was used.A solution of ferrous fumarate and βcyclodextrin in molar ratio 1:1 was mixed for 72 hours at room temperature, evaporated to reduce the volume and dried in a desiccator over concentrated sulfuric acid.Entrapment of ferrous fumarate in cyclodextrin was shown to increase the solubility of ferrous fumarate (Kapor et al., 2012).

Iron liposomes
Liposomes are produced by the association of amphiphilic compounds such as phospholipids into a bilayer structure using various techniques such as sonication, microfluidization, and solvent evaporation.Among these methods, microfluidization is the most promising technique in commercial scale production of liposomes.The encapsulation of food active ingredients in liposomes is still in its infant stages.However, interest in using this technique is becoming more popular.Liposomes are especially suitable for encapsulation of hydrophilic active components such as water soluble vitamins, minerals, and flavours.The encapsulation efficiency of active component depends mainly on the rigidity and the stability of the bilayer structure of liposomes (Matos et al., 2015;Gonnet et al., 2010).
A significant improvement in iron supplementation in animal trials was observed in liposomal encapsulated ferric ammonium citrate (Xu et al., 2014).They conclude that the liposomal iron can be used efficiently with minimal side effects to relieve the iron deficiency anemia caused by excessively exercise.A study that used rotary-evaporated film-ultrasonication method for the preparation of ferric citrate liposomes and heme liposomes demonstrated that this can be used as a supplement with iron fortified food in order to relieve iron deficiency.This study clearly shows that the liposomal iron delivery can significantly improve the iron absorption over direct iron supplements (Yuan et al., 2013a).Ding et al. (2009) synthesized ferrous glycinate nanoliposomes from egg phosphatidylcholine by the reverse phase evaporation technique with high encapsulation efficiency for oral administration.The prepared iron nanoliposomes showed controlled release potential and better stabilization under simulated gastric juice conditions .According to the findings by Yuan et al. (2013b), heme liposomes and ferric citrate liposomes demonstrated 119% and 54% increase in serum iron levels when compared to heme and ferric citrate, respectively.
It was found that ferrous sulphate liposomes prepared by reverse phase evaporation method functioned as a strong iron fortifier in fluid milk (Xia and Xu, 2005).The ferrous sulphate liposomes were created with egg phosphatidylcholine using thin-film, thin-film sonication, reverse-phase evaporation, and freeze thawing methods.Out of the four methods, reverse-phase evaporation was seen to create liposomes with the highest encapsulation efficiency (Xia and Xu, 2005).

CONCLUSION
Over the years, the method of iron delivery has evolved from using simple iron salts such as ferrous sulphate to chelates in the form of ferrous fumarate, ferrous bis-glycinate etc.With the advancement of research in carriers for delivery of small molecules, drugs, and nutrients, the use of proteins, alginate, and various hydrogenated oils have become promising candidates for iron delivery.Liposomal iron is no exception in showing that it is a good vehicle for iron delivery.The effect of these carriers on intestinal absorption of iron has indicated that the increase is probably due to the ability of the carrier to protect iron in the ferrous state.Therefore, for iron supplementation and fortification, the use of an effective carrier may be the method of choice.

Table 2 :
Estimated average iron requirement (Source: Institute of Medicine US Panel on Micronutrients).