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Iron deficiency anemia: A comprehensive review on iron absorption, bioavailability

and emerging food fortification approaches

Shubham Kumar, T. Anukiruthika, Sayantani Dutta, A.V. Kashyap, Jeyan A. Moses,

C. Anandharamakrishnan

PII: S0924-2244(19)30729-0
DOI: https://doi.org/10.1016/j.tifs.2020.02.021
Reference: TIFS 2756

To appear in: Trends in Food Science & Technology

Received Date: 30 August 2019

Revised Date: 24 January 2020
Accepted Date: 23 February 2020

Please cite this article as: Kumar, S., Anukiruthika, T., Dutta, S., Kashyap, A.V., Moses, J.A.,
Anandharamakrishnan, C., Iron deficiency anemia: A comprehensive review on iron absorption,
bioavailability and emerging food fortification approaches, Trends in Food Science & Technology (2020),
doi: https://doi.org/10.1016/j.tifs.2020.02.021.

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Iron deficiency anemia: a comprehensive review on iron absorption, bioavailability and
emerging food fortification approaches
Shubham Kumar, Anukiruthika T., Sayantani Dutta, Kashyap A.V.,
Jeyan A. Moses., Anandharamakrishnan C.*
Computational Modeling and Nanoscale Processing Unit
Indian Institute of Food Processing Technology (IIFPT)
Ministry of Food Processing Industries, Govt. of India, Thanjavur, Tamil Nadu, India.
*
E-mail: anandharamakrishnan@iifpt.edu.in

Abstract
Background
Anemia, a morbid condition, remains a global concern that affects people of all age groups.
This scenario attracts the attention of several government organizations for implementing
strict regulations to provide nutritional security. Iron fortification and supplementation has
been in practice from the past decades. However, there is a need for determining an effective
strategy to address this rising concern among the vulnerable population.

Scope and Approach

Among the existing approaches, iron fortification of foods remains to be cheaper and
effective in targeting large-scale population without the intervention of pharmaceutical drugs.
The key challenge is the bioavailability of iron from fortified foods. Thus, this work presents
a comprehensive review of morbidities of anemia, causes, the significance of haem and non-
haem iron, absorption, and bioavailability, in context with different iron fortification
approaches.

Key Findings
Apart from the nutritional deficit, anemia is also associated with a sedentary lifestyle linked
with obesity and diabetes. The complex interaction of elemental iron and its physiology have
been highlighted in consideration with potential iron enhancers and inhibitors. It was found
that the incorporation of haem iron would complement the effectiveness of non-haem iron
through fortification. Several iron fortification techniques focused on combating iron
deficiency have been described.

Conclusions
Food fortification remains to be a promising strategy for reducing the prevalence of anemia.
Food vehicles must be designed in considering its synergistic effects with iron complexes for
effective absorption and bioavailability. However, the scalability, cost economics, safety
concerns and acceptability of the iron-fortified foods remain as constraints that have to be
addressed. Further, the application of novel food processing techniques with food
fortification can result in the emergence of novel approaches for addressing iron deficiency
and anemia.

Keywords: Anemia; fortification; absorption; bioavailability; food vehicle.

1 1. Introduction
2 Anemia is a medical condition associated with increased or decreased RBCs characterized by
3 inadequate oxygen-carrying capacity to meet physiological needs (Marks, 2019). The most
4 common cause of anemia is iron deficiency; iron being an integral part of the blood protein,
5 hemoglobin. However, there are other abnormalities also related to anemia, such as deficiency of
6 vitamin B12, vitamin A, parasitic infections, chronic inflammation and inherited disorders
7 (Premkumar, Ramanan, & Thanka, 2018). Pregnant women and children are the most vulnerable.
8 At the global level, about 1.62 billion people are affected by anemia, corresponding to 24.8% of
9 the world’s population. In this cohort, the highest prevalence exists among preschool-aged
10 children (WHO, 2015). Estimates by WHO in 2011 explained that more than 40% of pregnant
11 women worldwide are anemic, associated with increased perinatal risks for mothers and
12 newborn. However, it was also reported that these women were already anemic at the time of
13 conception, with an estimate of 43% and 12% in non-pregnant women in developing and
14 developed countries, respectively. In such cohort, the prevalence of anemia becomes as high as
15 80 to 90%, when considering the WHO recommended Hb range of 120 g/L (WHO, 2015). An
16 interesting observation has been that iron deficiency is associated with a sedentary lifestyle.
17 Prolonged sedentary behavior leads to obesity. As observed, obesity and iron deficiency are
18 prevalent in low socio-economic groups that consume low-cost fast foods. Such foods have
19 lower nutrient levels and are rich in sugar and preservatives (Benotti et al., 2019). This scenario
20 is alarming and seeks the attention of governments and international bodies for implementing
21 effective nutritional policies and practices for the alleviation of this syndrome. Considering this,
22 the GNR of 2014 and the Micronutrient Forum 2014 Global Conference Proceedings emphasized
23 to organize health programs among the general public to highlight the significance of nutrition.
24 Although several techniques have been in practice for the prevention of anemia since the past
25 decades, there is a need for an effective strategical approach that run for the long-term. In this
26 context, iron fortification of foods remains a promising and cost-effective approach in targeting a
27 particular group of people in the long run. Various advancements and approaches are in progress;
28 for example, double and quadruple fortification is already in practice, providing the scope for the
29 delivery of more than one micronutrient. Often, foods such as rice, wheat, oil, salt, and tea are
30 selected as food vehicles for fortification as these are consumed in common irrespective of the
31 socioeconomic status of the population. The selected food carrier and the targeted micronutrient
32 must be synergistic as they have implications on the bioavailability of nutrients. Thus, for the
33 development of strategic approaches to address anemia through iron fortification, it is essential to
34 understand the fate of iron and its bioavailability. This work focuses on providing a
35 comprehensive review of causes of anemia, its classification, the nutritional significance of iron,
36 bioavailability, various iron fortifying approaches and food vehicles used for addressing iron
37 deficiency and anemia.
38
39 2. Overview of the existence of anemia
40 The World Bank estimates a value of USD 7 billion per year, over the next ten years, in addition
41 to existing resource allocations, to allow the world to reach World Health Assembly (WHA)
42 targets by 2025 for managing stunting, wasting, anemia in women, and increasing exclusive
43 breastfeeding. A report by WHO stated that a higher proportion of women become anemic
44 during pregnancy in both developed and developing countries. Worldwide, iron deficiency has
45 been considered a major form of malnutrition. This deficiency ranks 9th among 26 risk factors by
46 the global burden of disease 2000 project (GDB 2000).
47

48 2.1. Morbidities of anemia

49 The formation of oxy-hemoglobin by haem and oxygen present in the blood is significantly
50 disrupted due to anemia. This complex is essential for oxygen transport to muscles and tissues. It
51 is also an essential medium for various metabolisms in the human body (Conway & Henderson,
52 2019). It is vital to understand the widespread effects of anemia and its direct and indirect effects
53 on a country’s economy. The prevalence of anemia is unfortunately quite widespread, especially
54 in developing nations. The activity and productivity level of an average human being is affected
55 in anemia, reflecting on the productivity and economic traits of a nation at large.

56 In children, iron deficiency anemia (IDA) can cause a loss in weight and frequent respiratory and
57 intestinal infections. The most critical effect of anemia in children is impaired development in
58 behavior and psychomotor skills. When there is iron deficiency, the cell-mediated immunologic
59 response of T-lymphocytes is impaired. This, in turn, is due to reduced DNA synthesis that is, in
60 fact, dependent on the function of radionuclide reductase. The said enzyme requires iron for its
61 function (Aly, Fayed, Ismail, & Hakeem, 2018).
62 Similarly, in the case of adults, anemia has direct impacts on reduction in physical capacity; in
63 terms of percentage of GDP, physical productivity losses are greatest in developing countries
64 like India (1.18%) and Bangladesh (1.47%) where anemia and heavy manual labor are high. This
65 could be reversed by administrating iron therapy that can increase labor productivity by up to 5-
66 17%. Estimates showed that absolute losses of physical productivity due to iron deficiency are as
67 high as $4.2 billion annually in South Asia (Horton & Ross, 2003).
68
69 2.2. Prevalence of anemia: The Indian scenario
70 In India, more than 51% of women of reproductive age have anemia, and these figures have
71 increased from the 48% mark in 2016. Also, 38% of children (under 5 years) are affected by
72 stunning and 21% of this group are categorized under ‘wasted’ or ‘severely wasted’ (GNR,
73 2017). The prevalence of anemia in developing countries has had both direct and indirect effects
74 on the national economy (WHO, 2015). While anemia in most cases is acute, non-treatment can
75 make it chronic, or even life-threatening at a later stage.

76 A case study on the prevalence of anemia among the pre-school children in the southern part of
77 India showed that children of age below 24 months were more likely to have IDA than children
78 aged between 36-60 months, irrespective of gender difference. Another study reported by Rakesh
79 et al. (2019) showed a clear picture on the prevalence of iron deficiency and anemia among the
80 school children of 6-9th grades. Results estimated that the prevalence of anemia was around 44%,
81 among which 21.3% and 52.6% of the subjects did not have the habit of consuming leafy
82 vegetables and citrus fruits.

83 In another case study among adolescent girls in India, the prevalence of anemia was found to be
84 39% in adolescent girls, with severe, moderate and mild anemia accounting to 0.5%, 10.6%, and
85 27.9%, respectively (Premkumar et al., 2018). This grave situation is the effect of the poor
86 nutritional status of adolescent girls that has significant implications on their working capacity
87 besides, reproductive limitations. It must be taken into account that these deficiencies have to be
88 addressed at an early age, preferably early in adolescence, before marriage and childbearing,
89 through adequate iron-folic acid supplements, by means of medication or diet modifications.

90 These studies highlight the need for the implementation of intervention trials by the delivery of
91 iron through fortification or supplementation approaches. It is always advised to consume green
 92 leafy vegetables, mustard, green turnip, cereals, and sprouted pulses, eggs, roots and tubers
93 which are good sources of non-haem iron, and red meat, flesh, liver, and other viscera are rich
94 sources of haem iron, for improving the iron status of the body. Intake of iron-rich foods along
95 with folic acid, cyanocobalamin, and other B vitamins, ascorbic acid (vitamin C) is
96 recommended for improving the bioavailability of iron to prevent anemia. Foods such as
97 spinach, broccoli, fruits, liver, meat, eggs, milk are rich in folic acid and cyanocobalamin, while
98 citrus fruits such as orange, gooseberry, lemons are found to be rich sources of ascorbic acid.

99

100 3. Classification of anemia

101 Anemia is characterized by a reduction in hemoglobin (Hb) concentration, RBC count, or
102 packed-cell volume, and the subsequent functionality impairment of meeting oxygen demand in
103 tissues. The concentration of Hb in the blood greatly depends on physiological characteristics
104 like age, sex, and pregnancy status. Other factors that affect Hb concentration in the blood
105 include smoking habits, and environmental factors like high altitude area, to name a few. Thus, it
106 is vital to have insights on the various forms of anemia in order to better explain its prevalence
107 and to understand the causes of iron deficiency. Based on the morphology of RBC and the cause
108 associated with this condition, anemia can be classified into different types (Fig. 1).
109

110 3.1. Morphological classification

111 Based on the reticulocyte count, anemia can be classified as regenerative or hypo-regenerative.
112 For normal regenerative bone marrow, a decrease in Hb level shows an increased number of
113 reticulocytes that describe regenerative anemia; whereas, lack of regenerative capacity of bone
114 marrow cannot manifest the expected increase in reticulocytes causing hypo-regenerative anemia
115 (Sharourou et al., 2018). The mean corpuscular volume (MCV) also termed as average RBC
116 volume, groups anemia into microcytic (MCV<80 fL), normocytic (MCV 80-100 fL) and
117 macrocytic (MCV>100 fL) types. Microcytic anemia can be iron deficiency anemia [RBC
118 distribution width (RDW) >15%], thalassemia [RDW = 10-14%], and anemia of chronic
119 disorders. Iron deficiency caused by excessive bleeding is the primary reason for microcytic
120 anemia. Normocytic anemia is associated mainly with a nutritional deficiency (such as vitamin
121 B12, folic acid, and iron), renal failure and hemolytic anemia. Other causes include alcoholism,
122 use of drugs, radiotherapy, bone marrow infiltration, surgery, different infections and trauma. In
123 general, a pathologist does not consider macrocytic anemia as a disease. It is rather considered as
124 an RBC abnormality (Marks, 2019). Regular intake of alcohol, consumption of several drugs,
125 deficiency in vitamin B12 or folic acid is the major causes of macrocytic anemia, where unusually
126 large RBCs are formed with reduced Hb.

127

128 3.2. Classification based on erythrocyte production

129 Based on erythrocyte production, anemia is broadly categorized into two types. Decreased
130 erythrocyte production also referred to as ineffective erythropoiesis; this type of anemia results
131 from impaired red-cell proliferation that results in immature erythrocyte formation. The second
132 condition is the increased loss of erythrocytes because of hemolysis or due to severe blood loss.
133 In a few cases, anemia can occur owing to both the causes.
134

135 3.2.1. Nutrition deficiency anemia (non-haem)

136 The deficiency of nutrients and insufficient bioavailability of haemopoietic nutrients for
137 sufficient Hb concentration as well as for erythrocyte synthesis can result in nutritional anemia.
138 Bioavailability of haemopoietic nutrients such as iron, vitamin B12, folic acid, and ascorbic acid
139 get affected with contact with heat and light (Andlid, D’Aimmo, & Jastrebova, 2018). A range of
140 other factors such as polyphenols, cinnamon, phytates of whole grains and legumes, and calcium,
141 can also lower the bioavailability of elemental non-haem iron (Song, El Sheikha, & Hu, 2019).
142

143 3.2.2. Megaloblastic anemia

144 Usually, a deficiency of folic acid results in megaloblastic anemia. The inhibition of DNA
145 synthesis during RBC production leads to this anemic condition in which the cells are larger than
146 normal by size. This phenomenon is attributed to the inability of RBC to produce DNA quickly
147 enough to divide at the right time, and therefore the blood cells grow too large before division.
148 Several factors are responsible for this anemia, including vitamin B12 and folic acid deficiencies,
149 and even side effects of few chemotherapeutic or antimicrobial agents. Mainly, pregnant and
150 lactating women are more susceptible; therefore, it is recommended for them to consume
151 adequate amounts of folate and vitamin B12. This anemia can lead to pernicious anemia, which is
152 caused due to the lack of gastric intrinsic factors that are required for vitamin B12 absorption of
153 iron from food (Moll & Davis, 2017).
154

155 3.2.3. Sickle cell anemia

156 Sickle cell anemia is inherited as an autosomal recessive condition. Sickle-shaped cells formed in
157 this anemia block the blood flow through the spleen, causing splenic sequestration (Marks, 2019).
158

159 3.2.4. Iron deficiency anemia

160 IDA occurs when the total iron intake from foods or its absorption in the body is below the
161 recommended medical lower threshold. Low iron availability results in the medical condition
162 hypochromic microcytic anemia due to low haem concentration (Marks, 2019).

163 Anemia due to malnutrition or insufficient dietary intake is referred to as nutritional anemia. This
164 includes IDA, folate deficiency and, vitamin B12 deficiency. All of these can be addressed
165 through micronutrient supplementation and fortification strategies. On the other hand,
166 thalassemia and sickle cell occur due to genetic disorders, either alone or in combination with
167 IDA. These conditions can be addressed either by transfusion practices or by chelation therapy
168 that aid in targeting ineffective erythropoiesis and iron dysregulation (El-Beshlawy & El-
169 Ghamrawy, 2019). Table 1 summarizes the classes of anemia, remeady measures, and
170 intervention adopted to combat iron deficiency. In order to investigate the cause of deficiency, it
171 is essential to understand the mechanism of iron absorption and limitations, if any, that causes
172 the deficiency. This aspect of the syndrome has been discussed in the following section.
173

174 4. Mechanism of iron absorption and its inherent limitations

175 Iron transport in the digestive system is controlled by several iron-binding proteins such as
176 transferrin, lactoferrin, hemoglobin, and bacterioferritin, among other functional agents present
177 at several key sites. Generally, mucins get bound to iron in the acidic condition of the stomach
178 that helps in maintaining it in a solution state for later uptake in the alkaline conditions of the
179 duodenum. Mucin-bound iron can subsequently pass across the mucosal cell membrane. After
180 entering into cells, cytoplasmic iron-binding protein mobilferrin transports it to the basolateral
181 side where it is exported into the blood plasma. The mechanism of absorption of haem and non-
182 haem iron in the intestinal mucosal cells involve various transport process and regulatory
183 proteins as explained by Conway & Henderson (2019). In the human body, iron is absorbed in
184 both haem and non-haem forms, in different proportions. Hunt (2005) reported that the
185 absorption of haem iron from meat is most efficient and is generally unaffected by other dietary
186 factors, whereas absorption of non-haem iron is highly influenced by diet consumption patterns.

187 Ingested iron undergoes a series of complex changes in the human body (Fig. 2). As stated
188 earlier, dietary iron, absorbed through the intestinal mucosa, is present in two forms: haem and
189 non-haem. The former is better absorbed in the human body. Earlier, haem carrier protein-1
190 (HCP-1) was considered to be responsible for the absorption of iron into the duodenal
191 enterocyte. Later, it was proved that a folate transporter is responsible for this mechanism. Non-
192 haem (elemental) iron generally exists in two valence states: reduced ferrous (Fe2+) and oxidized
193 ferric (Fe3+) forms. Only the former can be absorbed by duodenal enterocytes. Thus, ferric iron
194 must first be reduced to its ferrous form by cytochrome b reductase (duodenal cytochrome b) or
195 other reducing agents present in the apical membrane of duodenal enterocytes. Later, it is
196 transported into the duodenal cytoplasm by the divalent metal iron transporter-1 (DMT-1).
197 Acidic conditions are required for uptake of elemental iron, as they aid in ferrous iron solubility
198 and supply protons for co-transport through DMT-1 (Conway & Henderson, 2019). Absorbed
199 ferrous iron can be utilized by the body in three different ways: (1) by being transferred to the
200 mitochondria for the production of haem molecules, (2) by being transferred into ferritin and
201 stored within the enterocyte, and (3) by being transported to other body sites where iron is
202 essential. Ferroportin, a transporter molecule found in the basolateral membrane and
203 reticuloendothelial macrophage, coordinates with ferroxidase hephaestin, and is responsible for
204 the circulation of ferrous iron (Helman, Anderson, & Frazer, 2019). Another ferroxidase
205 molecule is the ceruloplasmin of plasma, and it aids in iron-binding with transferrin in
206 extracellular fluids. Genetic deficiency of these proteins hinders the oxidation of iron that leads
207 to an overload of iron in the liver, pancreas, and brain. This condition could eventually lead to
208 neurological dysfunction and diabetes (Sharma et al., 2019). A small glycopeptide molecule,
209 hepcidin, released from the liver, helps in iron absorption and circulation. At enhanced levels,
210 hepcidin reduces iron absorption by retarding DMT-1 transcription at the enterocyte.
211 Accordingly, hepcidin causes iron sequestration, usually seen in chronic anemia and can be
212 downregulated by the rise in RBC production, iron deficiency, and hypoxia (Conway &
213 Henderson, 2019). Many studies that involves the investigation of the factors that influence the
214 iron absorption patterns are on-going. A recent study on the bioavailability of iron explores the
215 significance of dietary peptide molecules in a chelated form as a novel carrier to combat anemia
216 (Wu, Yang, Sun, Bao, & Lin, 2020). This study opens up a new way for improving the iron
217 absorption and bioavailability through food-derived iron chelated peptides which remain as a
218 basis for transformation of the existing food fortification approaches.

219

220 5. Nutritional and anti-nutritional enhancers or inhibitors of iron absorption

221 Ascorbic acid (vitamin C), folic acid, citric acid, peptides rich in amino acid cysteine, and
222 vitamin A are enhancers of iron absorption. The latter can prevent inhibitory effects resulting
223 from coffee, tea and phytates. Alcohol can increase the absorption of ferric iron, but not of
224 ferrous iron (Hallberg, Hoppe, Andersson, & Hulthén, 2003). While haem iron absorption is
225 reported to be enhanced by unidentified factors present in meat, fish, and poultry, non-haem iron
226 absorption can be enhanced by carotenes, retinoids, alcohol (by enhancing secretion of gastric
227 acid promoting valence state), citric, tartaric and malic acids (Moll & Davis, 2017). Navarrete et
228 al. (2002) reported a negative effect on the absorption of iron with minerals like Ca, P, Mg, and
229 other chemical compounds such as malonaldehyde, oxalic and phytates.

230

231 5.1. Ascorbic acid

232 Ascorbic acid increases iron absorption by reduction mechanisms involving ferric to ferrous
233 conversions, allowing its effective transport through microvilli of the duodenum. Generally, non-
234 haem iron absorption from food increases by incorporating meat and fish to the meal since the
235 incorporation of ascorbic acid reportedly complements such absorption. It is in fact efficient
236 enough to chelate iron that aids in the formation of a complex which remains soluble over an
237 extensive range of pH (2-11) (Habeych, van Kogelenberg, Sagalowicz, Michel, & Galaffu,
238 2016). Serna-Saldivar & Carrillo, (2019) reported a study on the effects of vitamin C on iron
239 absorption from maize and wheat. These researchers confirmed up to 84% and 48%, respectively
240 increase in the levels of iron absorption from these grains. Hardimarta, Yuniarti, and Aini (2018)
241 reported a study on the effectiveness of guava juice in increasing the erythrocyte index. Results
242 showed that consumption of guava juice increases the mean corpuscular hemoglobin
243 concentration (MCHC) by 3.99%. Another study concluded that ascorbic acid can counteract the
244 inhibiting effects of polyphenols on iron absorption (Villano et al., 2016).

245

246 5.2. Enhancers present in animal tissue

247 Studies reported that the addition of small amounts of pork meat to phytate rich meal can
248 increase iron absorption by up to 57%. In another study, a combination of amino acids similar to
249 that of fish was investigated with black beans; showing an approximately three-fold increase in
250 iron absorption. Similarly, the addition of meat powder (with or without ascorbic acid) can
251 increase iron absorption from weaning gruel by around 85% (Hallberg et al., 2003).

252 Several mechanisms that explain enhanced iron absorption by animal tissue have been proposed.
253 Literature claims that partially digested products from animal tissue possess the inherent ability
254 to bind iron via their histidine and cysteine residues, which can, in turn, enhance the absorption
255 (Hunt, 2005). Nevertheless, these mechanisms need to be better explained.

256

257 5.3. Phytates

258 The reduction of anti-nutrient metabolites is the most effective and reliable approach in
259 enhancing iron absorption. About 80% of phosphorus content of seed is in the form of phytates
260 and it is stored in globoids of certain plants such as soybeans. Though phytates inhibit iron
261 absorption, its role in combating kidney stones and enhancing the immune system cannot be
262 overloaded. Reportedly, phytates show contradictory results for iron absorption; with both
263 positive and negative inhibitions (Makowska, Zielińska-Dawidziak, Niedzielski, & Michalak,
264 2018). Ferric phytate is known to have low bioavailability. Studies showed that enzymatic
265 degradation of phytates using phytase alone cannot improve iron bioavailability in cereal flours.
266 Along with enzymatic treatment, hydrolysis of dietary fibers using xylanase and cellulose can
267 improve iron bioavailability. Phytate is known to be present at a high level in soy isolates. This
268 may be a reason for the inhibitory effects of soy isolates on iron bioavailability (Kusn &
269 Suyatma, 2017).
270 Conventional food processing approaches like soaking, malting, and fermenting can hydrolyze
271 phytate from its myo-inositol hexaphosphate form into intermediate myo-inositol phosphates,
272 thereby improving iron absorption from phytate-rich foods. Thus, higher phosphorylated
273 derivatives have a higher potential to inhibit iron absorption than less phosphorylated forms. As
274 stated earlier, phytates can be degraded by phytase either with native or added phytates during
275 the manufacturing process. It is reported that iron absorption can be enhanced by enzyme
276 treatment of phytates in soy products prepared using a phytase from Aspergillus niger (Song et
277 al., 2019). Phytase has also been used in other foods which are based on rice, maize, oat, and
278 wheat, to produce phytate-free foods. Iron absorption has even been increased by 12-fold, by
279 complete dephytinization of cereal-based foods. Lactic acid fermentation of cereal flours and
280 yeast fermentation during bread manufacturing can reduce phytate levels up to 80% and 50%,
281 respectively. It is also reported that iron absorption from wheat bread rolls was twice as that of
282 iron absorption from chapatti owing to yeast fermentation effects in bread (Hurrell, 2004).
283

284 5.4. Tannins

285 Reports confirm that tannins are responsible for lowering non-haem iron absorption even when
286 studied with iron absorption enhancer ascorbic acid. Few researchers have claimed that tannins
287 from tea inhibit iron absorption from cereal-based iron-fortified foods. They have even explained
288 that the formation of a complex in the intestinal lumen with tannins reduces the absorption of
289 iron. In this context, Delimont, Haub, and Lindshield (2017) reviewed the effect of tannin on
290 iron bioavailability. They reported that a single meal with tannins generally reduces iron
291 bioavailability, but prolonged use of tannin and consumption of condensed tannins from food
292 may not inhibit the iron status, based on results from animal model.

293

294 5.5. Polyphenols

295 Polyphenols are well known for inhibiting non-haem iron absorption from food. These include
296 flavones, flavanones, anthocyanidins, isoflavone, and isoflavanones (Teng & Chen, 2019). The
297 effect of fruit juice rich in polyphenols such as monomeric flavonoids on iron absorption was
298 studied by Villano et al. (2016). These authors argued about the dose-dependent inhibitory
299 effects of different polyphenols. Results showed that polyphenol-rich drinks have no significant
300 effects on the plasma hepcidin concentrations due to the counteracting effects of ascorbic acid in
301 the juice, thereby permitting better iron absorption. However, other studies showed an inverse
302 correlation between the polyphenol content of different vegetables and iron absorption. The
303 antagonistic as well as the synergistic effects of interaction among various phenolic compounds
304 are well reported (L. Chen, Lin, et al., 2019; L. Chen, Teng, & Cao, 2019).

305 Phytates and polyphenols inhibit both haem and non-haem iron components. Phytates,
306 phosphates, inositol, tannins, myricetins, polyphenols (such as flavonoids, catechins, gallic acid,
307 theaflavins, thearubigins), bran, dietary fiber, oxalates, calcium salts and hepcidin (regulator) and
308 excessive levels of cations such as zinc, magnesium, and manganese can also inhibit iron
309 absorption. However, an adequate amount of these bioactive compounds in the diet is essential
310 for achieving beneficiary anti-diabetic and anti-inflammatory effects (L. Chen, Gnanaraj,
311 Arulselvan, El-Seedi, & Teng, 2019; L. Chen, Lin, & Teng, 2020; L. Chen, Lu, El-Seedi, &
312 Teng, 2019). A recent report on flavonoids provided a piece of evidence for its anti-
313 inflammatory effects through the intracellular signaling mechanism of its chemical structure (L.
314 Chen, Lin, Xu, et al., 2020; L. Chen et al., 2018).

315
316 6. Iron bioavailability
317 6.1. Methods of estimation of bioavailability of iron in fortified foods
318 The bioavailability of different forms of iron can be determined by its valence state and
319 solubility. Based on solubility iron compounds can be grouped as (1) freely water-soluble (such
320 as ferrous lactate, ferrous gluconate, ferrous sulphate, and ferrous ammonium citrate); (2) poorly
321 water-soluble but soluble in dilute acids (such as ferrous fumarate, ferrous saccharate and ferrous
322 succinate); (3) water-insoluble and poorly soluble in dilute acids (such as elemental Fe powders,
323 ferric ammonium ortho-phosphate, ferric orthophosphate, and ferric pyrophosphate); and (4)
324 protected iron compounds (Fe EDTA) (Navarrete et al., 2002). Bioavailability of iron-based salts
325 in the human body follows the order: ferrous sulfate, ferrous lactate, ferrous fumarate, ferrous
326 succinate, ferrous glycine sulfate, ferrous glutamate, ferrous gluconate > ferrous citrate, ferrous
327 tartrate, ferrous pyrophosphate > ferric citrate, ferric sulfate (Hunt, 2005). Bioavailability can be
328 enhanced by chelation of iron using NaFeEDTA or ferrous glycinate which improves luminal
329 iron solubility and moderately influence inhibitory effects of phytates (Hunt, 2005). Key
330 approaches to evaluate the bioavailability of iron are explained below.

331

332 6.1.1 Chemical balance method

333 The chemical balance method of understanding iron bioavailability is an indicative measure of
334 the levels of iron retained in the body after food consumption. Differences between iron intake
335 and fecal iron levels are calculated by apparent iron absorption. Although this method provides
336 an idea about the characterization of mineral metabolism, it has limited use in the study of iron
337 bioavailability. This method is time consuming, insensitive and lacks precision owing to
338 challenges including incomplete fecal collection. This is because it can only be used to evaluate
339 bioavailability in the whole diet and not from individual meals. Also, it suffers from the
340 possibility of errors in calculating the balance of iron from intake and excretion values
341 (Fairweather-Tait, Phillips, Wortley, Harvey, & Glahn, 2007).
342

343 6.1.2. Solubility or dialyzability

344 Generally, for in vitro solubility studies, food samples are treated with HCl to adjust the pH to 2.
345 After this, the sample is digested with pepsin, followed by a change in pH to 6 and digestion with
346 pancreatin. Thus, soluble iron gets released and can be measured from the collected supernatant
347 (Stewart, Morton, Coad, & Pedley, 2019). On comparing iron bioavailability from processed and
348 unprocessed infant foods, solubility results showed that processed food has higher
349 bioavailability, but this was contradictory to the Hb status monitored through feeding trials.
350 Thus, with regard to the bioavailability of native iron, solubility is not an adequate indicator
351 (Fairweather-Tait et al., 2007). Iron dialyzability is considered as the improved approach of the
352 solubility method, where the levels of dialyzable iron are measured. NaHCO3 or 2, 2’ piperazine-
353 1,4-diylbisethanesulfonic acid (PIPES) buffer solution is generally used for this in vitro study.
354 Both dialyzability and solubility methods were evaluated by Kapsokefalou et al. (2005) in
355 different iron-fortified milk products and these researchers concluded that interaction of iron
356 compounds and the food affects the formation of dialyzable iron than its soluble forms. Thus,
357 dialyzability better explains iron bioavailability as compared to solubility (Fairweather-Tait et
358 al., 2007).
359

360 6.1.3. Caco-2 cell model

361 Caco-2 cells are human adenocarcinoma cells, widely used for the determination of iron
362 absorption. Caco-2 cell lines are distinguished and exhibit similar features of small intestinal
363 microvilli. For iron uptake studies, first, the sample is digested in in vitro condition, similar to
364 the solubility study. Then the digested food is exposed to cultured Caco-2 cells where a dialysis
365 membrane is placed, forming an upper chamber that protects the digestive enzymes similar to
366 that of the mucous layer as in the intestine. Iron from food samples gets dialyzed through the
367 membrane and becomes available for uptake by Caco-2 cells. Despite minor disagreements in the
368 effects of dietary factors on iron absorption, the Caco-2 cell model shows a good correlation with
369 human absorption patterns (He, Li, Ding, Li, & Li, 2019). Au and Reddy (2018) evaluated the
370 effect of different enhancers and inhibitors on the bioavailability of iron from a semi-purified
371 protein-enriched meal containing egg albumen. Similarly, the effect of different proteins like
372 casein, beef, bovine serum albumin and soy on iron bioavailability were analyzed using the
373 Caco-2 cell method. Relatively, fewer variations in soluble protein concentrations than with
374 different protein sources were observed with the addition of enhancers and inhibitors to egg
375 albumen. Data revealed a strong reduction in iron solubility and absorption in meals containing
376 phytate, tea, bran, casein and soy, which were consistent with human absorption studies.
377

378 6.1.4. Hemoglobin repletion method

379 Rat hemoglobin repletion method is a standard technique used by the Association of Official
380 Analytical Chemists’ (AOAC) (Franchini, Montagnana, & Lippi, 2010). This method is used for
381 determining the relative bioavailability value of iron, in comparison with ferrous sulphate. Male
382 rats are fed with the iron-deficient diet for a known period and later the rats are fed with iron-rich
383 diet (containing the iron compound to be tested) for a known period (Swain, Newman, & Hunt,
384 2003). During this study, individual rats are generally housed in wire bottom stainless steel cages
385 under a 12 h light-dark cycle. From blood samples drawn, variation in hemoglobin content
386 during the repletion period can be analyzed (Swain et al., 2003). The slope of the plot between
387 Hb and dietary iron concentration explains the quantitative measure of iron bioavailability. Thus,
388 the relative biological value (RBV) of iron sources understudy can be expressed against the
389 reference ferrous sulphate (Hurrell & Egli, 2019). This method can be employed for
390 investigating the efficiency of different food and non-food sources of iron in treating deficiency.
391 Apart from these methods, the bioavailability of iron has been studied using in-vitro cell culture
392 bioassays, in-vitro gastrointestinal studies, isotopic labeling, and using in-vivo methods.

393

394 6.2. Effect of dietary pattern on the bioavailability of iron

395 The extent of the bioavailability of the fortified iron varies with the iron compound used.
396 Valcárcel et al. (2019) performed an experiment to assess the effect of bioavailability of four
397 different iron compounds on the fortified cereals using pigs. The study employs ferrous sulphate
398 heptahydrate (FSH), ferrous fumarate (FF), electrolytic iron (EI) and micronized dispersible
399 ferric pyrophosphate (MDFP). Results showed that EI was less efficient in replenishing the
400 stored iron than other compounds. FF and MDFP resulted in equal bioavailability of iron that
401 significantly increased the iron stores of the weaned piglets. Thus, dietary patterns greatly
402 influence the distribution and absorption of dietary iron. In general, haem iron is better absorbed
403 (15-40%) than non-haem iron (1-15%). Reduction in meat consumption can decrease haem iron
404 levels (Hunt, 2005). The total amount of bioavailable iron from a mixed diet containing meat is
405 estimated to be 18%; whereas, the total amount of non-haem iron from a plant-based diet is
406 found to be only 10%. In such a scenario, a possibility of improving iron in vegetarian diets is
407 through fortification of iron in cereals and other plant-based products.

408 Key factors that affect the bioavailability of iron include the type and concentration of iron
409 fortificant, dietary factors, nutrient interactions and health status of the individual. The iron status
410 of an individual greatly influences the absorption pattern of non-haem iron while the effect on
411 haem iron seems to less significant. A recent study was reported on the effect of enhancement of
412 non-haem iron through the incorporation of haem iron in the diet. O’Flaherty, Tsermoula,
413 O’Neill, & O’Brien (2019) investigated the enhancing potential of bovine co-products in
414 improving the bioavailability of non-haem from infant rice cereals. Results showed that the
415 relative uptake of iron was found to be 207.13%, 171.21% and 265.28% for beef co-products
416 such as kidney, lung, and heart, respectively. These findings inferred that the iron uptake was
417 higher with the co-products than beef itself (30.23%), thus highlighted the potential function of
418 bovine co-products in improving the non-haem bioavailability from plant-based foods. Thus, a
419 wise combination of mixed diets can help in improving iron status and bioavailability. Futher,
420 the knowledge of iron enhancers and inhibitors must also be considered in the fortification of
421 foods for combating anemia.

422

423 7. Iron fortification of foods

424 Fortification is the common practice for increasing the content of an essential micronutrient like
425 vitamins and minerals in foods, so as to improve the nutritional quality of the food, and to
426 provide public health benefits at minimal risk (WHO, 2015).

427 The main aim of food fortification is to improve the nutritional content of the food. A major
428 criterion in deciding the best way of delivering micronutrients either by fortification or
429 supplementation depends on the target population group. Food fortification aids in easy access to
430 achieving daily nutritional needs for rural communities without dependency on pharmaceutical
431 supplements and has the potential to impact a large number of people in a cost-effective manner.
432 Iron supplementation involves the oral administration of pharmaceutical iron compounds while
433 fortification of iron involves the delivery of iron through foods. Supplementation of iron can be
434 practiced when immediate action is required for increasing the level of iron in the human body as
435 orally administered haem iron easily enters into the bloodstream. Whereas, iron fortification is
436 helpful for targeting a specific group by supplying iron through foods which follow the regular
437 absorption mechanism as that of the diet, resulting in a gradual increase of the iron status of the
438 population. In foods, iron fortification is done in order to meet the biological demand of iron, by
439 increasing the level of iron in the food significantly. Although the effect of fortification of foods
440 is not as quick as of supplementation in targeting nutrient deficiency, fortification is an effective
441 approach for sustainable benefits considering the long run (Khan, Singh, Upadhayay, Singh, &
442 Shah, 2019).

443 This review provides unambiguous evidence that iron concentration in the human bloodstream
444 can be improved through iron-fortified foods. Considering all age groups, the pooled positive
445 intervention can result in reductions in anemia and iron deficiency owing to the interference of
446 fortified iron from the food in the various pathways of iron absorption. Although food
447 fortification has several benefits, there exist certain limitations such as deterioration of the color
448 and flavor of food vehicles, determination of optimal delivery systems and the assurance of
449 health impact and acceptability of biofortified foods. However, fortification remains to be
450 attractive in terms of cost, since the costs of conventional medications remain high, but fortified
451 foods still remain harder to reach the poorest; those who are most price sensitive. Horton (2006)
452 reported a study on the economics of fortification and results showed that the effectiveness of
453 fortification in terms of cost ranges from $22 to $60/day, remaining comparatively favorable
454 than values of other health-care interventions for children. Another study on the benefits of
455 investment on iron fortification estimated a rise in benefit: cost ratio from 6:1 to 36:1, because of
456 combined effects of cognitive ability and physical productivity. On comparing food
457 supplementation, fortification, and dietary diversification, food fortification is known to be cost-
458 effective (cost of $66 per Disability-Adjusted Life Year - DALY), while supplementation and
459 dietary diversification had a cost of $179 and $103 per DALY, respectively (Detzel & Wieser,
460 2015). Therefore, for effective implementation of the fortification process, more studies are
461 required, considering public awareness and acceptability.

462

463 7.1. Food vehicles and iron fortification

464 One of the major requirements of food fortification is the need for proper identification and
465 suitability of a food vehicle. Essentially it is one that is commonly consumed by the target
466 population for high bioavailability of micronutrients (Hurrell & Egli, 2019). Another important
467 factor to be taken into account in selecting the food vehicle is the knowledge about chemical
468 interactions of food with iron compounds. Various criteria in the selection of food for
469 fortification include the dietary pattern, food habits, geographical regions, and socioeconomic
470 status. The most ideal characteristics of a food vehicle used for fortification include the focus on
471 the target population without making any significant changes in dietary patterns, but being
472 consumed regularly, enabling an increase in body iron levels (Detzel & Wieser, 2015). Common
473 foods that are ironfortified include salt, sugar, cereal-based products, milk, and other dairy
474 products (Navarrete et al., 2002). However, the potential effects of iron enhancers and inhibitors
475 have to be taken into account for increasing the effectiveness of fortification.

476 In this cohort, cereals and its flour as a staple food are more commonly used as food vehicles for
477 fortification. In conjunction, an estimate showed that the amount of fortification of flour was
478 found to be 97% in the US, 25% in Asia, 6% in Europe, 31% in Africa, 44% in Mediterranean
479 countries and 4% in Pacific regions (Cardoso, Fernandes, Gonzaléz-Paramás, Barros, & Ferreira,
480 2019). Rice, the staple food of Southern Asia, is biofortified through genetic engineering so as to
481 possess higher amounts of iron and beta carotene. Similarly, iron fortification of biscuits,
482 cookies, and bread are a common practice in the western world. Pulses hold an important part of
483 Indian diets, and fortification of iron in milled and polished split dhal and its flour would be a
484 cheaper vehicle for iron delivery. Other food vehicles include milk and fruit juices such as
485 orange, guava and grape juices. Studies were reported on iron fortification of foods using
486 cheaper food vehicles by considering the socioeconomic status of the Brazilian population. In an
487 8-month intervention trial, children who consumed iron fortified water (20 mg of Fe/ l) showed
488 significant reduction in anemic levels (Lamounier et al., 2010). In fact, each country has its own
489 choice of adopting a suitable food vehicle by taking into account culture, eating habits and
490 dietary patterns, income status of population groups and geographic regions.

491

492 7.2. Fortification approaches

493 Fortification focuses on increasing the content of one or more target micronutrients in the food
494 and this is predominantly done during food processing and value addition. On the other hand,
495 biofortification interventions happen at the farm-level; typically, through alterations in
496 agronomic practices, plant breeding or biotechnological approaches. Biofortification strategies
497 for iron fortification have the advantage of addressing large populations through advanced
498 biotechnological techniques. However, the adoption level of this approach is limited by
499 variations in crop yield, impact on agronomic factors and acceptance by farmers.

500

501 7.2.1. Biofortification

502 Biofortification is a biotechnological process focusing on macro and micronutrient aggregation
503 in plant cells. Several approaches such as (1) genetic modification; (2) transgenic approach; (3)
504 biofortification through agronomic practices, and (4) biofortification through plant growth-
505 promoting microorganisms have been explored. Biofortification through genetic modification
506 involves the modification of existing genetic traits for improvement or development of
507 micronutrients. This approach is widely adopted for improving the yield of crops with increased
508 nutritional content (Khan et al., 2019) (Table 2). On the other hand, the transgenic approach for
509 biofortification employs transfer of desired genes specific for micronutrient production from one
510 plant to another plant which is in lack of it. This can be accomplished through the insertion of
511 desired genetic traits responsible for trace element-binding proteins or though overexpression of
512 already existing storage proteins or by expression of storage proteins that are responsible for
513 uptake of micronutrients (Balk et al., 2019).

514 Agronomic biofortification involves the application of nutrient-rich fertilizers to foliage or roots
515 for increasing the concentration of micronutrients in plant tissues. In some instances where
516 mineral elements become unavailable from the soil, the direct application of soluble fertilizers on
517 foliar parts and roots is employed (Balk et al., 2019). Effectiveness of fertilizer on overall crop
518 growth and yield is greatly influenced by the type of fertilizer. Although soil contains adequate
519 amounts of iron, only a small portion is available to plants. An efficient way of delivering iron is
520 by using iron chelators. This is because conventional inorganic iron from the soil becomes
521 unavailable to plants because of adsorption, precipitation and oxidation reactions.

522 Biofortification of crops through plant growth-promoting microorganisms is considered to be a

523 promising supplementary approach in augmenting micronutrient concentrations besides
524 improving the yield and soil fertility. Generally, plants take iron from the soil present in the
525 oxidized form (Fe3+). Microorganisms such as bacteria and fungi have the tendency to produce
526 low molecular weight compounds (<10 kD), siderophores for sequestering iron compounds.
527 Plants' roots can take up siderophores which are secreted by plant growth-promoting
528 microorganisms, thereby enhancing iron uptake. Bacterial species such as Bacillus,
529 Pseudomonas, Arthrobacter, Enterobacter, Serratia, Azotobacter, Azospirillum and Rhizobium
530 and fungal species such as Aspergillus, Penicillium, and Rhizopus produce these compounds
531 (Khan et al., 2019).

532

533 7.2.1.1. Ferritin content enrichment

534 Ferritin, a principal iron storage protein in all living aerobic organisms, can store up to 4,500
535 Fe3+ atoms with good solubility and bioavailability. Ferritin is the complex of certain multi
536 proteins that are formed by the linkage of polypeptide chains of ferritin that are arranged
537 globular. Iron, in its native form, is converted to iron hydroxide, comprises of small micellar
538 units with large surface area, making it available for transport by reduction to Fe2+. When
539 attached to a protein molecule, apoferritin, the insoluble iron micelle, results in a complex stable
540 soluble molecule, ferritin. Ferritin comprises of multiple sub-unit protein shells surrounding an
541 inorganic iron-core in the form of microcrystalline particles. When the demand for synthesis of
542 Hb rises in the bone marrow, iron and amino acids of the liver are mobilized by the breakdown
543 of ferritin and other storage proteins (Chawla, Beers-Mulroy, & Tidmarsh, 2019). Hemosiderin,
544 another form of iron storage, is composed of variable granules with iron and organic
545 constituents, as well as some ferritin protein. This form of iron exists in the insoluble and
546 electron-dense form of ferric oxyhydroxide-phosphate micelles. Microscopy studies on
547 hemosiderin confirmed that hemosiderin is formed by the breakdown of ferritin, subsequent to
548 the loss of protein coats.

549 Increasing the iron storage capacity of grain endosperm through overexpression of ferritin is a
550 proven approach to improve the iron content of rice (Boonyaves, Wu, Gruissem, & Bhullar,
551 2017). Sabatier et al. (2017) evaluated iron absorption from iron-enriched yeast added to fresh
552 cheese. They reported 72-82% iron absorption from iron-enriched yeast indicating that yeast
553 lysed during digestion and released its iron. Makowska et al. (2018) reported a study on the
554 effect of extrusion conditions on the stability of iron and extruded product quality. Soybean
555 sprouts cultured in 20 mM FeSO4 were incorporated in corn snacks to supplement iron. Results
556 inferred that the iron stability of fortified snacks was greatly affected by moisture content.
557 Process conditions for ferritin fortified snacks were found to be 12% feed moisture with 140ºC
558 process temperature, yielding the desired expansion characteristics, with iron in its preserved
559 form (Makowska et al., 2018).

560

561 7.2.1.2. Increase in nicotinamine content

562 The iron homeostasis system in plants comprises a complex network of enzymes and Fe
563 chelators like citrate, deoxymugineic acid (DMA), and nicotianamine (NA), transporters and
564 other transcription factors. Naturally, plants take up iron (in Fe3+ form) from the soil. This form
565 of iron less soluble in the physiological pH range and combat this condition, plants of higher
566 order have evolved with two different strategies for iron uptake (Kobayashi, Nozoye, &
567 Nishizawa, 2019). Strategy I is the reduction-based mechanism involving the release of protons
568 from the roots to the rhizosphere, where iron from Fe3+ is reduced to Fe2+ and gets transported by
569 the Fe2+ transporter IRT-1 in non-gramineous plants. Strategy II occurs in gramineous plants
570 where iron uptake is enhanced by chelation-based mechanism, chelating Fe3+ through the release
571 of mugineic acid (MA) from the roots. This Fe3+-MA complex is taken up by the oligopeptide
572 transporter (YS1) and yellow stripe-like (YSL) transporters.

573 Nicotinamine (NA), a metal chelator is widely present in higher plants and acts as a key
574 component for assimilation of metals and homeostasis. Hence, by manipulating cellular NA
575 concentration, Fe concentrations in plants can be improved. NA, a non-proteinaceous amino
576 acid, helps in the synthesis of MA from L-methionine thereby involving in micronutrient
577 translocation in plants. It is proved that nicotianamine synthase (NAS) not only increases NA
578 production but also increases Fe and Zn levels in crops such as rice, soybean, and sweet potato.
579 Thus, overexpression of NAS through biofortification improves food quality and production
580 yield (Johnson et al., 2019).

581 Lee et al. (2009) showed that overexpression of the rice NA synthase gene (OsNAS3) results in
582 increased levels of iron in plants and NA content in seeds. Results have shown that anemic mice
583 fed with genetically modified seeds recovered to normal Hb and haemocrit levels within a two
584 weeks’ period, while those fed with wild type (WT) seeds remained anemic. Further, it was
585 proved that iron bioavailability can be increased by enhancing nicotianamine synthase (NAS)
586 expression in rice grains (Lee et al. 2009). Thus, NA enrichment approach of biofortification has
587 a good potential in combating iron deficiency.

588

589 7.2.2. Encapsulation

590 Encapsulation is a technique in which the bioactive component is entrapped into a matrix that
591 surrounds the active component thereby helps in the targeted and controlled release of the active
592 component (Fig. 3). In general, encapsulated forms of iron compounds are appropriate for dry
593 food ingredients and products like infant foods, dry beverage mixes, and minimally processed
594 foods. Examples of food fortified through encapsulation are presented in Table 3. Lipid-based
595 microcapsules help in preventing deterioration of color and also improves the bioavailability of
596 both iron and iodine from dual fortified salt. Ferrous sulfate and ferrous fumarate in their
597 encapsulated form are used for fortification of infant formulas and cereal flours from wheat and
598 maize. Research indicates that encapsulation of iron compounds with coatings of hydrogenated
599 oils from palm and soybean, mono and diglycerides and maltodextrins does not compromise
600 bioavailability (Hurrell, 2004).

601 Pre-treatment using non-thermal processing techniques like ultrasound can be used for the
602 incorporation of microencapsulated nutrients into food matrices. This treatment greatly promotes
603 the uniform distribution of iron content. The iron content of pumpkin was increased to more than
604 1000% as compared to control samples through ultrasound treatment (Rojas, Alvim, & Augusto,
605 2019). Abbasi and Azari (2011) reported a study on iron fortification of milk using
606 microencapsulation by liposome and fatty acid ester (FAE) methods. Results showed that the
607 liposome approach had higher iron encapsulation efficiency (85.5%) than the FAE method
608 (81.8%); FAE technique was found to be more simple, cost-effective and practically efficient for
609 iron fortification because milk fortified with the FAE method was similar to control samples in
610 terms of sensory characteristics (Abbasi & Azari, 2011). Cengiz et al. (2019) reported a similar
611 study on the oxidative stability of iron-fortified emulsions. They showed that ferrous sulphate
612 could be successfully incorporated into liposomes with higher encapsulation efficiency (89%).
613 However, challenges due to chemical reactivity due to oxidation of phospholipids need to be
614 addressed. In another study on liposomal encapsulation using ferrous glycinate, Ding et al.
615 (2009) reported that the stability of the iron compound can be significantly improved by
616 encapsulation using liposomes, by preventing disruption of ferrous glycinate from the
617 extracapsular lipid bilayer environment. Thus, ferrous glycinate liposomes are a promising iron
618 fortifier.

619 Gutierrez et al. (2016) reported a study on fortification of yoghurt using iron-entrapped
620 niosomes. In this study, niosomes were formulated with food-grade surfactants of glycerol
621 monoleate, polyglyceryl-3 dioleate or sorbitan monooleate and dodecanol were used as the
622 membrane stabilizer by adopting modified ethanol injection method. The encapsulation
623 efficiency was found to be in the range of 72-84%, with minimal effects on sensory attributes,
624 rheology, and stability of yoghurt fortified with iron-entrapped niosomes.
625 With the advancements in nanotechnology, it is possible to engineer ferritin nanoparticles that
626 can be available through physiological routes. Although these nano-sized particles have higher
627 bioavailability, the mechanism and action of synthesized ferritin mimetic nanoparticles are not
628 well understood (Blanco-Rojo & Vaquero, 2019). Nanoencapsulation is the technique of
629 encapsulating bioactive compound(s) inside a shell or a capsule. It involves incorporation,
630 dispersion, and absorption of bioactive compounds as small vesicles in the nano range (Vishali,
631 Monisha, Sundari, Moses, & Anandharamakrishnan, 2019). Nanoencapsulation not only
632 provides enhanced bioavailability but also provides stability, better handling and protection
633 against oxidation, controlled release and targeted delivery. Thus, it is proven as one of the
634 approaches for the fortification of iron. Hosny et al. (2015) reported a study on solid lipid
635 nanoparticles (SLNs) loaded with iron for treating - IDA. Hot homogenization and
636 ultrasonication approaches have been used for the preparation of iron solid lipid nanoparticles
637 (Fe-SLNs). Both in-vitro and in-vivo studies were done in rabbits. In-vitro results showed that
638 the entrapment efficiency using Fe-SLNs was found to be about 92.3%, with 25 nm average
639 particle size, while in-vivo results showed a four-fold enhancement in bioavailability. These
640 researchers concluded that Fe-SLNs could be a promising carrier for the fortification of iron with
641 enhanced oral bioavailability. Although encapsulation seems to limit the interaction of iron with
642 food matrices by making it more accessible for intestinal uptake, approaches using liposomes
643 and solid lipid nanoparticles are limited due to effects associated with iron oxidation (Cengiz et
644 al., 2019). Another major drawback is the deterioration of sensory properties and low
645 acceptability due to the addition of ferrous salts in encapsulated form (Siddique & Park, 2019).

646

647 7.2.3. Chelation and redox modulation

648 One of the concerns for the fortification of food with iron is the generation of undesirable
649 organoleptic properties of the food due to redox reactions and the formation of different
650 complexes which in turn decreases the acceptance of fortified foods. At low pH, iron remains as
651 Fe2+, whereas at high system redox potential (Eh), it gets oxidized to Fe3+. The state of iron
652 depends on the pH and Eh, following the Nernst equation (Eq.1) (Mehansho, 2006)
653 Eh = E0 + RT/nF ln [Fe3+]/[Fe2+] (1)
654 Where E0 is standard redox potential, n represents the number of electrons available in the
655 system, R is the universal gas constant (J/K.mol) and T is the temperature (K). To overcome this
656 issue, chelation-redox modulation has been proposed.

657 The chelation-redox modulation approach for iron fortification has been used for masking iron-
658 mediated metallic after-tastes through chelation. An amino acid chelated iron such as ferrous bis-
659 glycinate (a mole of ferrous iron chelated by two moles of glycine) is commonly used (Table 4).
660 Both pH and Eh are lowered by the addition of organic acids (such as citric acid) and reducing
661 agents (such as ascorbic acid) to create a reducing environment which prevents the development
662 of off-colors and effectively prevents oxidation of Fe2+ to Fe3+. However, stabilization of Fe2+
663 iron by redox modulation has its own limitation in applications to food systems with pH >5 as it
664 favors oxidation (Mehansho, 2006). Ferrous bis-glycinate when used for iron fortification,
665 without modulation, is oxidized to Fe3+. Redox modulation reportedly prevents this oxidation.
666 Among several chelating agents, ethylene di-amine tetra acetic acid (EDTA), a hexadentate
667 ligand that has six binding sites to the generic central atom, is commonly used.

668 To address these iron-mediated problems associated with food systems of higher pH,
669 encapsulation-based technologies have been used. Encapsulation involves making the iron
670 unreactive by isolating it from the food vehicle and other oxidizing agents using emulsifiers.
671 This approach works well for ferrous fumarate and ferrous succinate owing to their low-polar
672 nature, unlike other water-soluble iron sources such as ferrous sulfate, ferrous citrate and, ferrous
673 tartrate. When the ferrous fumarate-lecithin mixture is exposed to water it forms a bilayer. The
674 stabilization of ferrous salts depends on the bilayer formation by lecithin. However, more
675 attention has to be given for analyzing the toxic effects associated with the size of micronized
676 forms of iron.

677 Using chelated iron compounds, the iron status of the target population can be significantly
678 improved. Studies have been reported on iron fortification using NaFeEDTA to fortify fish sauce
679 and sugar. A similar approach was used to incorporate ferrous bisglycinate to dairy products
680 such as flavored milk and whey drink through which prevalence of anemia was drastically
681 reduced from 62.3% to 26.4% after one year in children and adolescents by regular consumption.
682 This approach was found to be effective as iron gets chelated in both NaFeEDTA and ferrous
683 bisglycinate, thereby protecting iron from reacting with other dietary components (Hurrell et al.,
684 2004).

685 Redox modulation and chelation using NaFeEDTA have the merit of formation of complexes
686 that aid in masking the metallic taste and prevention of color changes due to the addition of Fe
687 salts. If the extent of such complex formation is not well regulated, it may result in sourness and
688 darkening of food matrices (Habeych et al., 2016)).
689

690 7.2.4. Micronization

691 Iron salts like ferric pyrophosphate, ferric orthophosphate, and elemental iron powders are
692 generally inert in dry foods, providing the advantage of minimizing adverse chemical reactions
693 of changes in sensory properties and shelf-life. Nevertheless, these are associated with low iron
694 absorption in comparison with other salts such as ferrous sulfate. Thus, for improving
695 bioavailability, micronization and emulsification techniques have been used (Table 5). The
696 bioavailability of elemental iron powders in its pure form at zero valence state is related to the
697 size of the particle, surface area and solubility which differs with respect to its manufacturing
698 process (Hunt, 2005). Micronization is used to break ferric pyrophosphate in small parts to
699 solubilize it in food (Fig. 3). Though this process has greater future prospects, its high cost limits
700 the usage. The micronized form of ferric pyrophosphate having a diameter of 0.3-0.5 microns
701 has been developed recently for food fortification. The major advantage of this technique is that
702 it does not alter the chemical characteristics of food and/or its organoleptic properties.

703 Zimmermann et al. (2004) used a novel spray cooling for fortifying salt with iodine, iron and
704 vitamin A using hydrogenated palm oil for packing potassium iodate, micronized ferric
705 pyrophosphate and retinyl palmitate into microcapsules. The study concluded that incorporation
706 of the newly developed triple fortified salt in regular diets can significantly reduce the prevalence
707 of micronutrient deficiencies in school children with an average rise in total body iron to 105 mg
708 with 2% of fortified iron in a period of 10 months (Zimmermann et al., 2004). Roe et al. (2009)
709 reported a study on evaluating the relative bioavailability of SunActiveFe® (micronized
710 dispersible form of ferric pyrophosphate) and its suitability of incorporation into apple juice.
711 Results showed that iron absorption could be positively correlated with that of ferrous sulphate
712 and negatively correlated with serum ferritin concentration. Thus, the study proved that iron
713 bioavailability from the micronized formulation was higher than other ferrous salts, making it
714 potential fortificant for liquid food products (Roe et al., 2009).

715 Compared to other techniques, micronized ferric pyrophosphate (FPP) have no adverse effects
716 on sensory, color and textural properties of foods. However, this approach suffers low
717 bioavailability of iron, as compared to other fortification techniques (Haro-Vicente, Martinez-
718 Gracia, & Ros, 2006; Mashkour, Maghsoudlou, Kashaninejad, & Aalami, 2018).

719

720 8. Population intervention: combating anemia through iron fortification

721 Iron fortification of food is one of the interventions applied to combat iron deficiency. Fortifying
722 foods with iron would have a promising result in reducing the prevalence of anemia. A case
723 study was reported by Semba et al. (2010) investigating the intervening effects of iron-fortified
724 foods in lowering the risk of anemia among Indonesian children. Around 81,855 and 26,653
725 families of rural and urban areas, respectively were assessed during the study. Estimates showed
726 that the percentage of the existence of anemia among the children of the rural and urban families
727 were 55.9% and 60.8%, respectively. The study confirmed that administration of iron-fortified
728 milk and noodles among the subjects can result in reduced odds of child anemia in Indonesia.

729 In another study, the effectiveness of NaFeEDTA fortified soy sauce in controlling anemia
730 among the high-risk population of China was reported. Soy sauce was fortified with NaFeEDTA
731 at a concentration of 29.6 mg Fe/100 ml and was delivered to 14,000 residents of Bijie city.
732 Around 16 ml of iron-fortified soy sauce that provided 4.9 mg of iron was recommended for
733 daily consumption by each member of the family. Assessment of iron level was done by drawing
734 blood samples during the initial period and at 6, 12 and 18 months of all age groups of 3 to 6
735 years, 7 to 18 years, 19 to 54 years and > 55 years comprising of both males and females. The
736 consumption of iron-fortified soy sauce after the intervention of 18 months resulted in a
737 significant reduction of the prevalence of anemia among men, women, and children of all age
738 groups (Chen et al., 2005). Studies have also been reported on the enrichment of phytate rich
739 cereal flour with NaFeEDTA to improve iron bioavailability. Andango et al. (2007) compared
740 the effects of the electrolytic iron and NaFeEDTA in fortification of maize flour and assessed its
741 suitability for the reduction of IDA among the school children in Kenya. Porridge made from
742 whole maize flour fortified with NaFeEDTA at high (56 mg/kg) and low (28 mg/kg) doses and
743 electrolytic iron (56 mg/kg) were served five times a week to around 516 children of 3 to 8 years.
744 At the end of 5-month intervention trial, the concentration of Hb level was assessed. Results
745 showed that fortification with electrolytic iron does not improve the status of iron while with
746 NaFeEDTA a notable increment was observed (Andango et al., 2007). However, fortification
747 with a high dose of NaFeEDTA significantly decreased the prevalence of anemia than that of
748 low dose. These studies provide promising evidence for the intervention of anemia through iron
749 fortification of foods. Importantly, these approaches are population-specific and call for intensive
750 research and trials before mass implementation.

751

752 9. Challenges and constraints related to food fortification

753 Although iron fortification of staple foods and condiments remains to be a promising approach in
754 mitigating iron deficiency, concerns on overdosage of iron have to be considered cautiously.
755 Since there is no proven biological mechanism for the excretion of iron from the human body,
756 higher amounts of iron in the blood may have potential toxic effects. In humans, serum ferritin
757 levels of >20 µg/L is associated with poor absorption of haem and non-haem iron. Sometimes,
758 excess levels of iron can severe epidemic malaria as it favors conditions for pathogens. High iron
759 stores are one of the risk factors for cardiovascular disease, cancer, and diabetes mellitus
760 (Sharma et al., 2019). There emerges the risk of an increase in colonic iron in infants if iron
761 levels are increased through fortification. This can seriously influence microbiota equilibrium
762 and favor the growth of pathogenic bacteria than beneficial gut flora. Studies have been reported
763 that the addition of prebiotic galacto-oligosaccharides in infant formulae can effectively decrease
764 the adverse effects of iron in gut microflora (Paganini, Uyoga, & Zimmermann, 2016). Also,
765 multiple micronutrient supplementation in young children can result in the incidence of fever and
766 diarrhea. Sometimes, fortification of food with iron can lead to iron overload in persons with
767 hemochromatosis. Dietary sources such as black tea, turmeric, milk, non-citrus fruits which are
768 rich in natural iron chelators can help in declining adverse effects of iron overload. Thus, a
769 balance between iron uptake and utilization has to be regulated and monitored. This can be
770 addressed through effective implementation and enforcement of regulations. Monitoring of
771 additional intake of iron of non-targeted groups must be an integral part of any fortification
772 program.

773 Food Fortification initiatives estimate that fortification can significantly increase the price of the
774 commodity. For example, a price rise by 0.16% and 1.5-3% for 1 kg of cereal flour and 10 kg of
775 rice, respectively (from current retail prices), has been reported. The Food Safety and Standards
776 Authority of India (FSSAI) reported that fortification of wheat flour with iron, folic acid, and
777 vitamin B12 would hike 3-15 paisa per kg (Hoddinott, 2018). The burden of these costs can be
778 shared by multi-nutrient fortification, which could be a cost-effective approach. The cost
779 associated with each fortification approach would also differ; the cost of fortification through
780 encapsulation techniques can increase the price of the commodity by 2-5%. Benefit: cost ratio
781 calculations can be made by taking into account of the costs associated with nutrient
782 interventions, the prevalence of nutrient deficiency and its impacts on economic productivity.

783

784 10. Advancements and opportunities on fortification of foods

785 Precision nutrition is an emerging revolutionary approach to alleviate micronutrient deficiency
786 aiming in addressing individual differences based on physiological needs. This approach is based
787 on epigenetic modulation of nutrients in the diet through ‘nutrigenomics’. This flourishing field
788 can help in understanding how dietary components influence metabolic pathways and gene
789 expressions. In this line of omics technology, ‘nutriepigenetics’ refers to the influence of
790 nutrients in relation to gene expression without consequent changes in the DNA sequence, and
791 this can be exploited to address malnutrition. Another field of omics, ‘metagenomics’, refers to
792 interaction effects between diet, gut microflora, and associated metabolism. In the context of iron
793 fortification, these technologies can aid in the modulation of genes that are responsible for iron
794 metabolism (Blanco-Rojo & Vaquero, 2019). Thus, iron regulation through hepcidin can be
795 effectively monitored by these novel approaches.

796 Further, precision nutrition not only helps in assessing the physiological requirements of iron but
797 can also be used as an effective tool for grouping people based on body’s iron status. This
798 assessment helps in a faster and easier segregation of anemia-vulnerable-population, that would
799 ultimately result in the targeted recommendations of iron-rich diets. However, different criteria
800 such as dietary patterns, physiological response and genetical phenotypes must be taken into
801 account for the implementation of precise nutrition. The ‘omics’ sciences provide a promising
802 solution in customization of iron-fortified foods whilst considering public health. With the
803 advancements of these technology, the computational data of the above-mentioned factors could
804 be gathered for the development of food databases. These data driven models could provide
805 useful information in predicting the prevalence of anemia among the population groups, thereby
806 providing insights for the design of novel interventional approaches to combat anemia through
807 iron fortification of foods. The development of these novel technologies could pave a way for the
808 transformation of conventional interventional approaches and the emergence of precision
809 nutrition.

810 Nachal et al. (2019) have presented an interesting review on the applications of 3D food printing
811 in food processing, highlighting the scope of 3D food printing techniques to provide personalized
812 nutrition. The printing material supply can be tailored in terms of nutritional requirements of the
813 population. The personalized food formula can have blends of enhancers of iron absorption,
814 apart from other key ingredients for a healthy diet. Such approaches can go very well in localized
815 population groups and can prove the societal-wellbeing focus of food customization techniques.

816

817 11. Directions for future work

818 Recent advancements in biotechnology would open up for a new paradigm in areas of
819 computational genomics relating with the iron requirement and associated biophysics at the
820 molecular level. This would help in the biofortification of iron through bioengineering of plant
821 and animal tissues. More sophisticated analytical approaches using nanotechnology maneuver in
822 addressing the iron deficiency through food fortification by considering the dosages and toxicity.
823 The application of artificial intelligence and robotics in food engineering will lighten up the
824 emergence of novel interventions for iron fortification of foods.

825 Importantly, in the context of food fortification, the problem of anemia should be looked from
826 the regional perspective. This can bring about significant overall changes at the national and
827 international level. The issue can be better addressed with the identification of region-specific
828 diets that can be prospective food vehicles, research and sustainable approaches for food
829 fortification, novel food formulations with strong considerations on enhancers and inhibitors of
830 iron absorption, and more importantly, by creating widespread awareness on the role of healthy
831 diets and the significance of food micronutrients. Being a topic of global interest, research on
832 iron fortification must consider cost:benefit aspects. Importantly, research findings must have the
833 potential for mass-adoption and must be strongly linked to policies on nutritional security.

834

835 12. Conclusions

836 Although iron supplementation has a large impact on health than a fortification, iron fortification
837 would cost lesser and has the major advantage of aligning to the functioning and physiology of
838 the human body. This, fortification remains to be the safest intervention to address the concern of
839 iron deficiency. Various technical opportunities like safety and efficacy of nanotechnology in
840 food fortification are to be investigated for its long-term applicability. There is a need for a better
841 understanding of bioavailability of the nutrients and to overcome organoleptic and degradation
842 problems associated with the fortificants. Most efforts to combat iron deficiency using
843 fortification strategies have so far been concentrated in overcoming technical problems like
844 discoloration, off-flavor development and fatty acid oxidation, often neglecting the consideration
845 of other practical problems such as large-scale production, marketing, and quality control which
846 are crucial for successful implementation.

847 There remains a need for the development of novel food carrier systems that permit correlation
848 with food processing techniques. To achieve this, a deeper understanding of dietary components,
849 processing methods, and physiological factors are essential. This must then be linked to the
850 chemodynamics of iron during food processing and after ingestion, as this critically decides the
851 fate of the mineral during processing and digestion. Challenges associated with the stability of
852 iron compounds, powder/product characteristics, toxicity, and sustainability also need to be
853 addressed for individual applications.

854
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1151 (2019). Development of Micronutrient Fortified Extruded Rice Analogues. European
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1158 https://doi.org/10.1093/ajcn/80.5.1283
Table 1. Remedy measures for anemia and iron deficiency
Anemia Type Causes Remedy Recommended Interventional
foods practices
Nutritional deficiency Normocytic Due to the lack of Intake of foods Green leafy Dietary supplement or
anemia iron, protein, vitamin rich in non-haem vegetables, spinach, fortification
B12 and B9 iron and B broccoli, beans,
vitamin lentils and flesh
foods
Megaloblastic anemia Macrocytic Occurs due to folate Intake of folate Eggs, red meat, Oral/ intravenous folic
and vitamin B12 fortified cereals
and vitamin B12 acid supplement or iron
rich foods
deficiency fortification
Iron deficiency anemia Microcytic Results due to chronic Intake of foods Spinach, liver, Dietary supplement or
blood loss rich in haem iron sardines, poultry fortification
Sickle cell anemia Hemolytic Genetical disorder that Intake of calcium Colored fruits and Oral supplementation/
results due to the and iron rich vegetables, food Fortification/ blood
mutation of gene foods grains, calcium transfusion (if needed)
responsible for fortified tofu, soy
abnormal hemoglobin milk and orange
juice
Thalassemia Microcytic and Genetical disorder due Intake of iron rich Red meat, green Folic acid supplements/
hemolytic to the mutation of beta foods with leafy vegetables, fortification/ iron

40
 globin gene vitamin C iron fortified cereals chelation therapy/ blood
transfusion
Aplastic anemia Macrocytic Occurs due to an Intake of iron rich Beef, chicken, liver, Blood transfusion/ bone
insufficient production foods essential for fish, tofu, spinach marrow transplant
of RBC hemoglobin and and other leafy
RBC production vegetables
Pernicious anemia Macrocytic Autoimmune disorder Intake of vitamin Baked sweet potato Oral/ intravenous
that occurs due to lack B12 rich foods with peel, injections
of intrinsic factor watermelon,
results in impaired avocado, beet,
uptake of vitamin B12 pomegranate, beef,
nuts and seeds
Sideroblastic anemia Microcytic/ Associated with Intake of foods Liver, sea foods, Oral supplementation/
normocytic/ myelodysplastic rich in copper and mushroom, soy blood transfusion/ bone
macrocytic syndrome vitamin B6 bean, eggs, whole marrow or liver
grain cereals transplant

41
Table 2. Examples of recent studies on iron fortification of foods through biofortification

Biofortification Food vehicle Study parameters Key inferences References

approach
Ferritin enrichment Cassava Plants were engineered to IRT1 + FER1 plants could provide an estimated average Narayanan et
co-express mutated A. requirement of 40-50% and 60-70% for iron and zinc al. (2019)
thaliana iron transporter respectively in 1 to 6-year-old children and nonlactating,
(IRT1) and A. thaliana nonpregnant women
ferritin (FER1)
Ferritin enrichment Beans Study evaluated the iron Iron absorption ratio from common bean Pontal, targeted for Nutti et al.
bioavailability of common biofortification and compared with common bean BRS (2018)
bean BRS Pontal, targeted Estilo was not significantly different
for biofortification and
was compared with
common bean BRS Estilo
Increment in Rice Study evaluated the All single insertion transgenic lines had significant increase Boonyaves et
nicotianamine and efficacies of the in iron concentration, both in polished and unpolished al. (2017)
ferritin content MsENOD12B and native grains. The concerted expression of AtIRT1, AtNAS1, and
AtIRT1 promoters for the PvFER was a more effective strategy in achieving the
expression of AtIRT1 in highest iron increase of up to 10.46 mg/g dry weight

42
 rice in both types of gene
cassettes, and found that
native AtIRT1 promoter
to be a better choice for
driving the AtIRT1
expression in
biofortification.
Nicotianamine Rice The impact of contrasting Transgenic approach resulted in increase in the DMA Rodriguez-
enrichment levels of 2´- concentration alone; whereas, in combination with NA the Menendez et
deoxymugineic acid prevalent mechanisms appear to be those based on Fe(III)- al. (2018)
(DMA) and NA on iron DMA, which enhance Fe transport and storage in the
distribution in the embryo endosperm, likely using yellow stripe 1-like (YSL)
and endosperm of rice transporters (first and second scenario).
seeds has been assessed When increase in DMA occur in combination with NA
using wild type rice and increases, an additional mechanism based on Zn(II)-NA
six different transgenic appears to be elicited, boosting Zn transport and storage in
lines overexpressing the endosperm.
nicotianamine synthase
(OsNAS1) and/or barley
nicotianamine amino
transferase (HvNAATb)

43
Nicotianamine Wheat Constitutive expression An increase in bioavailability of iron in white flour milled Johnson et al.
enrichment (CE) of the rice from field-grown CE-OsNAS2 grain. This was positively (2019)
nicotianamine synthase 2 correlated with NA and DMA concentrations
(OsNAS2) gene was
engineered in bread wheat
to upregulate biosynthesis
of nicotianamine (NA)
and 2′-deoxymugineic
acid (DMA)
Phytic acid reduction Traditional NaFeEDTA - Fe Reduction in phytates levels from 6.2 to 4.6 mg/g when P. Glahn et
meal with Fe concentration of 2800 fortified lentil was added, thereby reducing the PA:Fe molar al. (2018)
fortified µg/g ratio from 8.8 to 0.9. Such effect was due to dephytinization
lentil dhal of fortified lentil during the fortification process.
Ferrous sulphate Wheat Ferrous sulfate (3–15 Pasta at 4% (Fe 0.026 mg/g) in iron-deficient diet fed to Naveen
(abiotic stress mM) were used to fortify anemic rats for 2 weeks had percentage iron absorption Kumar,
treatment) the wheat flour at 0.1 mg (PIA) and hemoglobin regeneration efficiency (HRE) of Muthukumar,
Fe/g 85.3% and 44.4%, respectively, higher than NaFeEDTA. and
Prabhasankar
(2019)

44
Table 3. Examples of recent studies on encapsulation for iron fortification of foods

Fortification Food Study parameters Key inferences References

approach vehicle
Encapsulation of Cheddar Compared effect of iron Iron-fortified cheese had higher hardness and gumminess Siddique and
Ferrous sulphate cheese fortification on scores than control cheese due to proteolysis of the protein Park (2019)
microstructural, matrix and binding with iron crystals during storage. On
textural, and sensory comparing, control cheese had higher sensory scores than
characteristics of the iron-fortified cheeses, and microencapsulated cheese
caprine milk Cheddar had the lowest scores for all tested sensory properties.
cheese.
Microencapsulation Milk 10 mg/L - C. Gupta,
of iron by liposome powder Chawla, and
method + ferrous Arora (2015)
sulphate
Encapsulation Milk 1 mg/g - C. Gupta,
FAE (fatty acid powder Chawla, and
ester) method + Arora (2015)
FeSO4 + PGMS
(polyglycerol
monostearate)
Encapsulation Milk 60 mg Fine in colour and no off-test raised C. Gupta,

45
(microcapsules were powder Chawla, and
prepared using SA, Arora (2015)
pectin and modified
starch) by freeze-
drying method +
FeSO4 + ascorbic
acid
Encapsulation Milk 0.798 mg - C. Gupta,
emulsification powder Chawla, and
method + SA Arora (2015)
(sodium alginate)
Encapsulation Orange juice 18 mg/500 ml Fruit juice fortified with iron pyrophosphate improves iron Blanco-Rojo et
Iron pyrophosphate status by increasing transferrin to saturation level after 8 al. (2011)
(Microencapsulation) weeks; haematocrit, MCV and Hb levels increased after 12
weeks
Microencapsulation - Drink 20 ppm of iron and 100 Lipid oxidation was significantly slower in encapsulated Kim et al. (2003)
polyglycerol yogurt ppm of vitamin C iron than in uncapsulated iron fortified yogurt. In terms of
monostearate sensory, no significant effects were found in
(coating) microcapsulated iron and vitamin C fortified drink yogurt
(FeNH4(SO4)2.4H2O) during 20 days of storage.
and L-ascorbic acid.

46
(core)

Microencapsulation Salt Ferrous sulphate (10% Comparing with Eudragit EPO, chitosan was an effective Veronica Dueik
of iron using spray and 20% pay loads of coating agent with 10 µm microparticles upto 25% loadings & Diosady
drying total solids) (2017)
Colloidal process Yoghurt 1150 ppm Fe3+ Results of lipid oxidation revealed that the delivery of Naghibalhossaini
Iron loaded double transition metals using double emulsions requires improved et al. (2018)
emulsion strategies to ensure efficient trapping of the metal ions
within the inner aqueous phase to prevent direct contact
with vulnerable substrates.
Colloidal process Soya based 8 mg/100 g Relatively stable during storage in terms of organoleptic, Kusn and
Ferrous sulphate, infant microbiological and physicochemical qualities. Suyatma (2017)
ferrous fumarate, and cereals
ferric pyrophosphate
Colloidal process Fish oil 4 mg of iron (Fe3+) /100 Initial iron encapsulation within the internal aqueous phase Choi, Decker,
Multiple emulsion g emulsion of the water-in-oil emulsions was as high as >99.75% and McClements
although a small amount leaked out over 7 days storage (10 (2009)
µg/100 g emulsion) and when water-in-oil-in-water
emulsion were mixed with fish oil droplets, the formation
of thiobarbituric acid-reactive substances decreased

47
Table 4. Examples of recent studies on iron fortification through chelation and redox modulation

Fortification Food Study parameters Key inferences References

approach vehicle
Redox modulation Lentil dhal Study aimed to NaFeEDTA was the most suitable fortificant for red lentil Taran et al.
FeSO4.7H2O, identify appropriate dal at 1600 ppm, providing 13–14 mg of additional Fe per (2017)
NaFeEDTA and methods and optimal 100 g of dal. Also, lentil dal sprayed with fortificant
FeSO4H2O dosage for Fe solutions, followed by shaking and drying at 75ºC
fortification of lentil performed best with respect to drying time and colour
dal change.
Redox modulation Tea Fortified tea was Molar ratio of 1:2 Fe:EDTA was able to avoid complex Dueik, Chen, and
EDTA prepared from premix formation and provide 4 mg of iron per cup of brewed tea. Diosady (2017)
by spraying iron and
sodium EDTA into tea
leaves. Iron
concentration in tea
was adjusted to 5
mg/cup
Redox modulation Bouillon 2·5 mg stable Fe Results indicated that Fe bioavailability in humans Cercamondi et al.
Ferric pyrophosphate cubes isotopes/cube increased by 46% as compared to bouillons fortified with (2016)
only (FePP) or FePP FePP only (4·4%) and bouillons fortified with FePP + NaPP
+ tetra sodium (6·4 %). Fe absorption from bouillons fortified with FeSO4

48
pyrophosphate only and with FeSO4 + NaPP was 33·8 and 27·8%,
(NaPP); ferrous respectively.
sulphate (FeSO4)
only, or FeSO4 +
NaPP
Redox modulation Banana Monosodium citrate Although colour change and metallic taste was reduced, Habeych et al.
Citrate was chosen as puree from 0.7 to 6.5 g/100 sourness was introduced in banana puree (2016)
ligand g banana
Redox modulation Rice Ultra-Rice formulated Enhancement of soluble iron was affordable by low-level Johns et al.
NaFeEDTA with NaFeEDTA at 2– NaFeEDTA or Na2EDTA.2H2O supplementation suggested (2015)
7% of total iron, or that this fortification strategy may merit further studies as a
with Na2EDTA.2H2O means of increasing absorbable iron in ultra-rice.
at 0.5X the total iron
equivalents increased
soluble iron by 7-
30fold
Redox modulation Tempeh 24 ppm Organoleptic test showed no differences between tempehs Sudargo et al.
NaFeEDTA (2013)
Redox modulation Powdered 4.8 mg/ 200 g - Mehansho (2006)
Ferrous bis-glycinate fruit
beverage

49
Redox modulation Fish sauce 10 mg/ 10 ml Regular consumption of iron-fortified fish sauce Thuy et al.
NaFeEDTA significantly reduced the prevalence of iron deficiency in (2005)
Vietnamese women within 6 months intervention
Redox modulation Ground Triple fortification: Around 80.8% of iron (ferrous bisglycinate chelate), 75.4% Soares et al.
ferrous bisglycinate roasted 4.2 mg of iron, 2.1 mg of (2019)
chelate, ferrous coffee of zinc and 300.0 mg zinc (zinc lactate), and 72.1% of calcium (calcium lactate)
sulfate, ferrous of calcium per 100 g were recovered in the expresso brewed coffee
fumarate of ground roasted
coffee
Redox modulation Yoghurt Around 75 mg iron Addition of iron salts does not change the yoghurt’s Ziena & Nasser
Iron amino acid amino acid characteristics. Among the four salts, yoghurt fortified with (2019)
chelate, ferrous chelate,160 mg ferrous amino acid chelate was highly prefered
sulfate, ferrous sulfate, 65 mg ferrous
fumarate, and ferric fumarate, 65 mg ferric
hydroxide poly hydroxide poly
maltose maltose was taken that
provides 20 mg Fe/Kg
milk

50
Table 5. Examples of recent studies on micronization approach for iron fortification of foods

Fortification Food Study parameters Key inferences References

approach vehicle
Micronization Potato Whole potato was Results indicated that vacuum impregnation and Mashkour et al.
Micronized Ferric fortified with iron by ultrasound assisted vacuum impregnation processes (2018)
pyrophosphate and using vacuum significantly enhanced the iron content of about 137.5
elemental ferrous impregnation with an and 210%, respectively. Relative bioavailability of
sulfate ultrasound pre- fortified cooked potatoes using micronized forms of iron
treatment. Whole potato was lower than the ferrous sulfate, but it had no
tubers were placed into undesirable effect on sensory evaluation, colour
a 5 L vacuum chamber parameters, and texture of fortified potatoes.
containing 1% (w/w)
iron solution (mass ratio
of the solution to potato
of 6% w/w)
Micronization Infant cereal 120 mg Fe/kg was fed to Both FF and MDFP were equally as bioavailable as the Caballero
Ferrous sulfate foods weaned piglets reference salt, and were used significantly better than EI Valcarcel et al.
heptahydrate (FSH), in piglets. These results contribute to extend the (2018)
electrolytic iron (EI), evidence-based results for recommending the most
ferrous fumarate (FF), suitable fortificant for infant cereals
micronized

51
dispersible ferric
pyrophosphate
(MDFP)
Micronization Ultra Rice® Study investigated the Although FeNaEDTA and ferrous fumarate resulted in Li, Diosady, and
Ferric-sodium EDTA stability of multiple darker colored grains, they had a much higher in vitro Jankowski (2008)
(FeNaEDTA), ferrous fortified Ultra Rice® bioavailability than formulations containing FePP.
fumarate, ferric formulations to develop
pyrophosphate a stable premix
(FePP), and containing iron, zinc,
SunActive-Fe® and B vitamins
Micronization Citric juice 49.2 mg Fe/l of juice Percentage of dialyzable total iron from ferrous bis- Haro-Vicente,
Ferrous sulphate, (pineapple glycinate (31.0%) was statistically higher than those Martinez-Gracia,
micronized and passion from ferrous sulphate (28.4%) and micronized ferric and Ros (2006)
dispersible ferric fruits) pyrophosphate (28.2%).
pyrophosphate and
ferrous bis-glycinate
Micronization Rice 6.34 mg of iron Double fortified rice analogues found to possess 99.85% Yogeshwari,
Ferric pyrophosphate analogues (micronised ferric and 99.7% of iron and zinc retention respectively Hemalatha,
from broken pyrophosphate) and 2.10 Vanniarajan,
rice flour mg of zinc (zinc oxide) Saravanakumar,
per 100 g of broken rice &

52
 flour Kavithapushpam
(2019)
Micronization Mixed fruits 3.2 mg Fe per 200 mL 15% RDA of Fe in 200 mL serving of juice fortified with Fleige, Bhoite,
Ferric pyrophosphate and of beverage ascorbic acid resulted in good Marwaha, &
vegetable Fe bioavailability for this form of Fe Glahn (2018)
beverage

53
Figure 1. Classification of anemia

54
Figure 2. Schematic representation of mechanism of iron absorption

55
Figure 3. Encapsulation and micronization approaches for iron fortification

56
Highlights
• Iron deficiency anemia remains as a major threat to global health
• Iron absorption and bioavailability depend on the delivery vehicle being used
• Role of inhibitors/ enhancers must be considered during fortification/ supplementation
• Food fortification is a promising approach to improve the iron status
• Nevertheless, research needs are high, and heavily population-specific