Iron Supplementation Influence on the Gut Microbiota and Probiotic Intake Effect in Iron Deficiency—A Literature-Based Review

Iron deficiency in the human body is a global issue with an impact on more than two billion individuals worldwide. The most important functions ensured by adequate amounts of iron in the body are related to transport and storage of oxygen, electron transfer, mediation of oxidation-reduction reactions, synthesis of hormones, the replication of DNA, cell cycle restoration and control, fixation of nitrogen, and antioxidant effects. In the case of iron deficiency, even marginal insufficiencies may impair the proper functionality of the human body. On the other hand, an excess in iron concentration has a major impact on the gut microbiota composition. There are several non-genetic causes that lead to iron deficiencies, and thus, several approaches in their treatment. The most common methods are related to food fortifications and supplements. In this review, following a summary of iron metabolism and its health implications, we analyzed the scientific literature for the influence of iron fortification and supplementation on the gut microbiome and the effect of probiotics, prebiotics, and/or synbiotics in iron absorption and availability for the organism.


Introduction
Iron deficiency is a prevalent problem affecting over two billion individuals around the globe. The main causes of this insufficiency are the lack of iron-rich foods in the diet or the body's inability to absorb ingested iron owing to acquired or genetic causes. Iron deficiency affects about 40% of the population in developing countries and around 10% of the inhabitants of developed countries [1]. Iron can be obtained through food products, supplements, or medical procedures such as blood transfusion and/or transfusion of red cell concentrates [2]. The most significant concern is not the source of the iron, but the type of iron obtained and its absorption pathways [3][4][5]. Foods such as vegetables, meats, and meat products are proven to be an excellent source of iron, and the fermented version of these products improves dietary iron absorption. Lactic acid, produced by lactobacilli, increases the dietary bioavailability of iron, thus suggesting that utilization of probiotic bacteria can act as a clinical tool for iron bioavailability optimization [6][7][8]. Environmental oxidized iron has low solubility, and for this reason, organisms must develop adequate modalities to compete for the available sources. Human growth and wellbeing are directly proportional to the consumption of this nutrient and are highly affected by the gut microbiota composition [9][10][11]. An increasing number of studies connect iron bioavailability and absorption to microorganism activity in the gut [12][13][14]. Furthermore, the virulence of many pathogens is associated with iron levels [15]. Solely 10% of dietary iron is absorbed in the human body, with the remaining 90% being excreted through fecal matter, thus affecting microbiota balance [16].
In this review, we analyze the scientific literature concerning the influence of iron fortification and supplementation on the gut microbiome and the effect of probiotics, prebiotics, and/or synbiotics in iron absorption and availability for the organism.

Iron Metabolism: Implications in Health and Disease
Owing to its implication in metabolic processes, iron is essential for all organisms. Its most studied functions are those linked to transport and storage of oxygen, electron transfer, synthesis of hormones, DNA replication, cell cycle control, nitrogen fixation, and antioxidant properties [10,17]. The amount of iron in the body of an adult human male is 3.5-4.0 g, with most of it being found in hemoglobin (Hb) [3]. Iron transport in the digestive system is controlled by several iron-binding proteins (e.g., transferrin, lactoferrin, and bacterioferritin), among other functional agents present at several key sites. Generally, mucins bind to iron owing to the acidic conditions in the stomach, which helps in maintaining it in a soluble state for later uptake in the alkaline conditions of the duodenum. Mucin-bound iron can subsequently pass across the mucosal cell membrane. After entering inside the cells, mobilferrin, the cytoplasmic iron-binding protein, transports it to the basolateral side, where it is exported into the blood plasma. The mechanism of absorption of hem and non-hem iron in the intestinal mucosal cells involves various transport processes and regulatory proteins ( Figure 1) [18]. It is at this stage that ferritin levels are considered as the most reliable markers of iron deficiency [17]. Overview of intestinal iron absorption and metabolism involving enterocytes, hepatocytes, macrophages, and erythroid marrow-adapted from [19][20][21][22][23][24][25][26][27].
Legend: Synthesis of Heme and other metallo-proteins requires >20 mg of iron/day (from diet and mostly by recycling); from this, only 1-2 mg of iron is derived from intestinal absorption (mainly in the duodenum). Heme iron from the diet is readily absorbed (through a Heme transporter) and non-Heme iron is predominantly found as ferric iron (Fe 3+ ). To facilitate the transport of insoluble ferric iron across the luminal membrane of enterocytes, ferric iron (Fe 3+ ) is reduced by the enzyme ferric reductase duodenal cytochrome B (DCYTB) to ferrous iron (Fe 2+ ), which is then transported into the enterocyte through the divalent-metal transporter (DMT) 1. Iron's secretion into the circulation from the basolateral membrane of enterocytes and macrophages uses ferroportin (FPN). A ferroxidase, hephaestin, is also involved in the export process by converting Fe 2+ back to Fe 3+ . This step is required for the attachment of iron to apo-transferrin, thus forming transferrin in plasma. This di-ferric transferrin is the form in which iron is delivered to sites of iron utilization, such as the erythroid marrow, where the red blood cells (RBCs) are produced. FPN is controlled by hepcidin (from hepatocytes). Binding of hepcidin to FPN causes the latter protein to be internalized and degraded within the enterocyte. Hepcidin, when found in high amounts, inhibits iron release by ferroportin. However, when it is low in concentration, it facilitates iron export into the circulation. In a few cell types (enterocytes, macrophages) iron can be stored as bound to ferritin. In the circulation, iron, which is toxic in the unbounded state, is attached to apo-transferrin (as mentioned above), forming plasma transferrin.
Anemia manifests itself thanks to an inadequate diet or poor iron absorption. An iron deficiency can cause anemia, resulting in the inability to maintain proper tissue oxygenation and body homeostasis [28,29]. About 52% of pregnant women, mainly in developing countries, may develop this deficit. Iron requirements are increased during pregnancy, because of increased necessities; for example, a normal enlargement of red blood cell (rich in hemoglobin) diameter can be observed [3,30]. Regarding the prevalence of iron deficiency anemia (IDA), the World Health Organization (WHO) reports that about 50% of all anemia cases are caused by iron deficiency [31]. A classification of anemia (based on red blood cell morphology) is presented in Figure 2. Other classifications are based on criteria such as erythrocytes' production, nutrition deficiencies (iron, proteins, folate, vitamin B12, and vitamin B9), genetic deficiencies in synthesis of Hb chains, red blood cells cytoskeleton with abnormal shapes, or based on secondary pathologies including chronic bleeding [18]. In some cases, sedentary lifestyles, diabetes, and obesity are also linked to anemia [18,33,34].
According to WHO [31], anemia caused by iron deficiency can be classified according to three stages, ranging from the first (mild form) to the third stage (severe anemia) with low Hb (<70 g/L for children under 5 years and pregnant women, and <80 g/L in children over 5 years, adolescents, and adults). This last type will affect the cognitive and physical functions of the individual, resulting in the patients presenting chronic fatigue and lower work capacity [9,35]. However, it can be corrected through diet or with iron-based treatment. The most common causes of iron deficiency are low solubility of iron, low bioavailability, and loss of blood due to hemorrhage [6]. The explanatory factor for these conditions is that most of the iron from ingested food has low solubility and, consequently, low bioavailability [1]. Once absorbed, iron enters into the systemic circulation and becomes available either for storage or for the physiological functioning of the body [36].

Gut Microbiota and Iron Deficiency
The human gastrointestinal microbiota has an essential role in metabolism and cognition, thus influencing vital functions of the human body. The balance in our diet and nutrients influences the healthy growth of the microbiota in the first years of life [37][38][39][40]. In order to modulate the gut microbiota balance and avoid a dysbiosis, probiotics and prebiotics can be administered orally, by fecal microbiota transplant or by other methods [37]. Probiotics are live microorganisms that, once introduced in the gut, improve its microbial composition. Prebiotics are selective non-fermented food ingredients that bring changes in the microbial activity by feeding the probiotics, and thus providing many benefits to the host [41].
The intestinal microbiota of adults and children differs depending on several factors such as age, ethnicity, geography, and lifestyle [38]. A healthy diet that contains whole grains, fresh fruits and vegetables, nuts, and medicinal foods helps us fight various diseases and maintain a proper balance of the gut microbes [11,42]. Although physiological and neurological consequences of iron deficiencies have been addressed extensively, further studies need to evaluate their impact on development of gut microbiota [43,44]. The absorption of iron, which can result either from diet or oral supplements, is a complex mechanism containing many reactions. The literature surrounding mucosal iron absorption [45][46][47][48] is controversial in the debate of iron absorption, presenting unclear explanations of whether it is absorbed in the ferrous or ferric form. However, it was agreed that the key role of iron absorption is the intestinal solubility of the oral medication [49]. According to a study, during the weaning process of different babies from Africa, the gut flora was changed by the fortification of iron, having as consequences a reduction in the number of Bifidobacteria as well as an increase in the Enterobacteriaceae and some specific enteropathogens (e.g., E. coli pathogenic). Moreover, in the same study, it was mentioned that the fortification of iron increased the faecal calprotectin levels, showing the inflammation of the intestine [34]. It is established that, when the iron levels drop, intestinal infections can occur because of the influence on the gut microbiota composition [50]. A study conducted on young women in south India correlated a low iron level with low levels of lactobacilli in the feces [51].
Both deficiency and excess of iron levels are important in terms of gut microbiota dysbiosis and the appearance and development of inflammation and colorectal cancer [42,52,53]. There are specific bacteria that mediate the development of intestinal pathologies such as Streptococcus bovis, Enterococcus faecalis, and Clostridia. They release hydrogen sulfide and secondary biliary salts, which accelerate inflammation and carcinogenesis [54,55]. Some bacteria, including Bifidobacterium longum and Lactobacillus acidophilus, prevent intestinal inflammation [56]. According to [57], gut inflammation was not influenced by the high amount of additional iron (50 mg Fe, 4 days/week) for 266 days and the concentration of the main microorganisms and short chain fatty acids (produced by the probiotic species) in the fecal matter was unchanged. A previous study, conducted on rats by the same authors, showed that iron-deficient rats had considerable lower concentrations of butyrate and propionate (produced specially by Roseburia spp./E. rectale group) in the cecum and the gut microbiota composition was strongly modified [58]. This implies that iron deficiencies have a notable impact on the gut microbiota, while iron excess does not.
It was determined that the alteration in a host's iron homeostasis may affect "the luminal iron content of the intestine, and thus the composition of intestinal bacteria" [59]. An analysis of mice feces demonstrates that the iron regulatory protein 2 (Irp2) and the protein of the mutated genes that appears in diseases such as hereditary hemochromatosis Hfe (affected in hereditary hemochromatosis) are highly involved in iron regulation. The test results demonstrated that, compared with the wild type mice control, the microbiota composition has significantly changed in mutant mice (Irp2-/-or Hfe-/-). Therefore, the iron metabolism in the host highly affects the type of host gut microbiota [59]. Moreover, the pH of the colon contents is a factor that dictates iron absorption. For example, microorganisms can ferment galacto-oligosaccharides, leading to a decrease of the pH in the intestine, and thus facilitating iron absorption at this level. Therefore, the acetic acid containing products made by probiotics can be introduced into the diet, and could improve iron absorption [60].
According to a study, fluctuations of iron concentrations can have pathological effects, negatively influencing the intestinal microbiota composition. Compared with germ-free mice colonized with gut bacteria, in germ-free mice, an 8-to 10-fold increase in metal transporter protein 1 (DMT 1) and DCYTB expression and a twofold reduction in ferroportin expression in the duodenum can be observed. The enterocytes of germ-free mice were low in iron and, after colonization with gut bacteria, the epithelial cells favorized iron storage [61]. The mechanism is explained in a study conducted by Gonzales et al. 2017 [62]. They demonstrated that Lactobacillus fermentum, a main probiotic found in human microbiota, exhibits a remarkable ferric-reducing activity. P-hydroxyphenyllactic acid, a metabolite produced by this strain, increases iron absorption through the DMT 1 transporters from enterocytes by reducing the Fe 3+ to Fe 2+ . Similar results can be found in a study (on Kenyan infants) that showed how iron-fortified powder can negatively influence the gut microbiota composition by increasing the number of the pathogens, thus causing intestinal inflammation [34].
In terms of genetics, different types of hereditary hemochromatosis are caused by mutations in the HFE, hepcidin (HAMP), hemojuvelin (HJV), ferroportin (SLC40)A1, and transferrin receptor 2 (TfR2) genes that correspond to genetic disorders of iron overload [63,64]. Ferroportin is an exporter of hepcidin, the hormone that regulates iron in the body, which is affected by mutations in the HAMP gene [65,66]. Intestinal epithelial and fecal metal contents are directly influenced when a lack of proper function of ferroportin occurs because this protein is involved in iron export into circulation from both enterocytes and macrophages [67]. Mutations in iron regulatory gene SLC40A1, which encodes ferroportin, cause a hepatic iron overload hemochromatosis phenotype that negatively affects the intestinal microbiota [68,69].
An iron-rich environment in the gut is conducive to Proteobacteria. Studies in children have shown that excess iron causes inflammation and the growth of pathogenic bacteria [34,70].
Inflammatory bowel disease leads to a lack of iron absorption. Iron excess from the intestinal environment can create pathobionts (potentially pathological cells) that are a result of the transformation of commensal microbiota bacteria [71]. Colorectal cancer may be induced by these pathobionts, which can also induce inflammatory bowel disease [72].

Iron Absorption from Supplements and from Foods
Although iron is an essential nutrient in our diet, iron deficiency is the most frequent nutritional disorder worldwide. Iron availability, in heme or non-heme form, in various foods such as legumes, milk, meat, and juice, was demonstrated many years ago [73]. Iron absorption in the intestine occurs by different pathways depending on the type of iron present. Absorption of heme iron (Fe 2+ ), predominantly found in the meat diet, is partially known and probably mediated by a heme carrier into the enterocytes. In the cells, iron is released from heme by hemoxygenase-1 (HO-1). Non-heme iron (Fe 3+ ), predominantly present in the vegetarian diet, is reduced by ferric reductase CYBRD1 (DCYTB) enzyme to Fe 2+ before being transported into the intestinal absorptive cells. Once non-heme iron is reduced to heme iron in the intestinal lumen, the latter is transported into the intestinal enterocyte by a protein called the divalent metal transporter (DMT 1). Inside the enterocytes, iron can be either stored as ferritin or transported through the basolateral membrane into the bloodstream by ferroportin (FPN1), also called the iron exporter. Hepcidin regulates the FPN1 expression. Hephaestin modulates the metabolism and homeostasis for iron absorption, namely by integrating two Fe 3+ into one transferrin molecule (Tf) and linking iron with apo-transferrin.
There are many options regarding the treatment of iron insufficiency, including oral iron derivates, which can effectively get absorbed in the intestine and mostly in duodenum, taking into consideration that the stomach and the other parts of the gut are less involved in absorption processes [49].
It has been shown that adding meat and juice to legumes increases the availability of iron because vegetables contain protein and minerals, but are limited in ascorbic acid. Milk had the lowest iron absorption rate owing to a large amount of calcium present. In order to increase the iron absorption, pregnant women and children should consume foods with as much ascorbic acid as possible, but not simultaneously with milk [74]. In a study, mice were tested by giving them water, green tea, black tea, or tea extract while consuming iron-based diets. The results showed that both black and green tea do not pose a risk with respect to the availability of iron and do not adversely affect the growth or metabolism of rats, but the interaction of iron absorption in humans with these tea compounds should be studied more closely [75]. Iron accumulates in the body from the first days of life, and when there are no genetic defects, its absence from the body signifies the presence of unhealthy foods in the first months of life or in the mother's diet. It has been reported that ironfortified formulas are beneficial in preventing this deficiency. There are studies on newborns (up to maximum 32 weeks of age) that have followed the two categories of nutrition: those who received iron fortified formulas and those fed with their mother's milk. Iron retention was calculated every week (Hb). Statistics have seen a balance of iron in the body as hemoglobin from birth and hemoglobin from the first 3-6 months was not considerably changed between infants fed with milk formula and those fed with human milk [17].
There are several microelements and metabolites, such as ascorbic acid (vitamin C), folic acid, citric acid, peptides rich in the amino acid cysteine, and vitamin A, that enhance intestinal iron absorption [76][77][78][79]. Some of these molecules can act indirectly, by abolishment of the inhibitory action of phytates found in coffee and tea [80]. Polyphenols (anthocyanidins, flavones, flavanones, isoflavones, and isoflavanones) inhibit the non-heme iron absorption from food. Alcohol simultaneously inhibits the ferrous iron absorption and increases the absorption of ferric iron [18]. It is also shown that the administration of probiotics and prebiotics may increase iron absorption [6,60,81,82].
Iron deficiency anemia impacts people of all ages worldwide. Thanks to the severity and high prevalence of this problem, governmental organizations implemented strict regulations to ensure proper iron nutritional needs. Fortification of foods with iron has been a common practiced in recent decades. However, the greatest concern is the physiological availability of iron ingested from fortified foods. Foods, acting as iron carriers, must be tailored considering their synergistic influence with iron complexes for absorption and increased availability. However, in this approach, there are several issues that need to be solved in terms of large-scale applications, economic efficiency, safety, and consumer acceptability [18].
Iron fortification is a process that includes the addition of vitamins and minerals, which are essential micronutrients used to improve the food quality from a nutritional perspective. To overcome iron deficiency anemia, affected individuals should consume foods that are rich in micronutrients. Microorganisms developed the siderophores (high-affinity iron-chelating compounds, secreted by microorganisms) system, a mechanism for obtaining iron from the environment. However, the excess of iron affects microorganisms' pathogenicity; thus, to minimalize the negative effect of iron fortification, a better approach should be created. Moreover, iron is absorbed in the presence of fermentable carbohydrates that stimulate "the growth of bacteria that produces short-chain fatty acid such as propionic acid", which increases mineral intake [83]. Micronutrient deficiencies are mostly observed in impoverished populations. A possible solution for this problem could be the supplementation of micronutrients to increase the nutritional status. An example of such supplementation is multiple micronutrient food supplements [52], which include vitamins such as B1, B2, B6, B12, and A, as well as calcium, iron, and lysine. "Ferrous sulfate is the most bioavailable form of iron" [59], and its interactions with micronutrients can lead to an increase of absorption.
The main foods used for iron fortification are cereals and dairy products and, to a smaller extent, salt, sugar, and condiments. Using cereals, their flours, and derived food products as iron carriers is disadvantageous because of their high phytic acid content, which can diminish iron absorption [1]. Unabsorbed iron destroys the gut microbiota and causes changes in the ratio of protective and pathogenic bacteria. It is thus recommended to treat this deficiency intravenously [56].
Fortified iron foods using probiotics could help reduce anemia. This technique has several advantages because it is less costly, more accessible, and reduces the use of medicines. By developing this emerging approach for mitigating micronutrient deficiency, precision nutrition can be achieved. [74]. Precision nutrition helps group people according to the iron content in their body, thus allowing the recommendation of targeted diets that are rich in iron, leading to a decrease of anemia frequency in the population [13].
For food scientists, the greatest and most important challenge is to provide food for the growing population, while considering the content of macro and micronutrients. Hence, several procedures need to be tailored to properly increase the iron content and reduce inhibitors in food crops. Moreover, it is necessary to study the effect of some food additives, such as prebiotics, probiotics, and metal chelators [36].

Probiotics and Iron Deficiency Treatment
Considering that the hazard of iron deficiency for the worldwide population is of high importance, it is crucial to implement appropriate strategies to combat this problem. The most frequent strategies are nutrition programs; iron supplementation in foods; iron drug supplements; and probiotic, prebiotic, and symbiotics approaches [30,33,60,73].
A systematic review demonstrated how the use of Lactobacillus plantarum 299v helps in the prevention of iron deficiency anemia. It has been found that this probiotic improves dietary nonheme iron absorption in active Caucasian Europeans [6]. Rosen et al. [81] also uses L. plantarum 299v for the treatment of iron deficiency in pediatric patients, but with no favorable results for iron absorption. Low-dose ferrous sulfate (1-3 mg/kg/day) was administrated to children, with or without the probiotic. The researchers observed no significant difference regarding serum ferritin level in subjects taking the probiotic L. plantarum 299v when compared with controls. No association was made regarding ferritin level and probiotic use.
Another study, performed on rats, investigated the effects of oral multispecies probiotic (Bifidobacterium bifidum W23, B. lactis W51, B. lactis W52, Lactobacillus acidophilus W37, L. brevis W63, L. casei W56, L. salivarius W24, Lactococcus lactis W19, and Lc. lactis W58 in equal proportions), administrated in low 2.5 × 10 9 and high 1 × 10 10 dosages. The result revealed that a single log increase in the dosage influences the total counts of probiotics in the feces. Concerning the iron-binding capacity, it was higher in the high dose probiotic group when compared with the control group. Both high and low dose probiotic groups manifested low levels of serum iron, thus high iron levels in the liver, pancreas, and duodenum were registered in the high dosage probiotic groups [82].
Body inflammation alters nutrient's availability, including iron, as a mechanism for microbial growth limitation. One of the leading causes of acute gastroenteritis is Salmonella enterica serovar typhimurium (S. typhimurium). Recent studies have shown that S. typhimurium flourishes in the inflamed gut while hunting for iron siderophores. By administering a probiotic strain, Escherichia coli strain Nissle 1917, that uses similar mechanisms to assimilate iron, S. typhimurium colonization in mouse models was reduced. E. coli Nissle probiotic activity hangs on the iron acquisition, given that mutants lacking in iron uptake colonize the intestine, but do not reduce S. typhimurium counts in the feces. Iron availability influences S. typhimurium colonization and E. coli Nissle reduces S. typhimurium intestinal growth by competing for this restricting nutrient [84]. Table 1 shows most relevant studies related to iron absorption in relation to single or multiple probiotic strains administration. Table 1. Probiotic intake in different types of iron deficiencies and their effect on the iron level in the organism. IDA, iron deficiency anemia.

Streptococcus thermophilus
Orally; fermented milk beverage ↑ iron absorption and utilization (amelioration of blood hemoglobin, serum iron, total iron binding capacity, ferritin) [87] Anemia (IDA) L. fermentum Orally; nanoparticles probiotic internalize into the enterocyte delivering the nanoparticles and providing an adequate iron level [88] Anemia (IDA) L. fermentum In vitro ↑ iron absorption [62] In menstruation L. plantarum 299v Orally; capsules, with a meal with a high iron bioavailability ↑ iron absorption when administered together [89] Anemia (IDA) L. plantarum 299v Orally; fruit drink ↑iron absorption . [7] Anemia (IDA) L. acidophilus Orally; fermented bread ↑ ferritin formation significantly in the intestinal cells (in vitro) and animal serum (in vivo) ↑ iron absorption [90] Anemia (IDA) L. plantarum 299v Orally; capsules together with iron and vitamin C ↑ iron level in the blood [91] Low iron bioavailability No difference on the iron status, height, weight, and gut microbiota profile [33] "↑"-increase;

Prebiotics and Synbiotics in Iron Deficiency Treatment
Prebiotics are functional food components that stimulate the growth and colonization of beneficial bacteria in the gut, and ultimately improve the health of the body. A noteworthy role in intestinal physiology is played by gut microbiota colonization. To effectively reduce the risk of certain diseases (cancer, lipidemia, and so on), we can take advantage of prebiotics such as galactooligosaccharides, fructo-oligosaccharide, inulin, and pectin. These components help to implement proper gut microbiota colonization and to maintain its balance [41]. Pre-and probiotic consumption is called symbiosis.
Probiotic fructans (selective carbon sources) fortify the health of the hosts. They influence blood parameters, such as decreased blood urea, uric acid, ammonia, and nitrogen balance. Several studies that have been conducted claim that fructans such as inulin have beneficial effects on the body's colon functions, even improving minerals' absorption. There are two types of physiological effects: direct effects within the large intestine and on the intestinal microbiota and indirect systemic effects that influence the metabolism and reduce the risks of disease [94].
Several studies correlated prebiotic and/or synbiotic intake to an increase of iron availability, mostly by converting Fe 3+ to Fe 2+ owing to their ferric-reducing activity, and promoting iron uptake by enterocytes. Most of these studies are summarized in Table 2. Anemia (IDA) inulin, polidextrose, arabic gum, and guar gum.
Orally in yoghurt, 2 g per day ↑ heme iron bioavailability not influence non-heme iron bioavailability [107] *CIDCA -Centro de Investigación y Desarrollo en Criotecnología de Alimentos ** EDTA -Ethylenediaminetetraacetic acid In one study, pectin was assembled to iron nanoparticles as matrices for delivery of L. plantarum CIDCA 83114. Scientists investigated the biophysical stability of iron-pectin nanoparticles and analyzed their effect. The results showed that iron is not toxic for the probiotic cells, and did not affect the bacteria's viability, which means that it is efficient in both iron delivery and bacterial stabilization. This can also be used as an alternative solution to overcome iron deficiency [101]. An experiment on Sprague-Dawley rats showed that the intake of non-purified prebiotic galacto-oligosaccharides for 3-4 weeks might enhance Ca, Mg, and Fe absorption [108].
Another study aimed to synthesize organic compound derivatives from polysaccharides ready to bind iron to help in iron deficiency treatment. An inulin-succinic anhydride-cysteine-ferric chloride complex was obtained, having good biodegradability in the presence of inulinase and high muco adhesion properties. The results revealed that the potential employment of such complexes in the oral treatment of iron deficiency anemia or as a supplement of iron-fortified foods can be utilized [109].
A study revealed that synbiotic treatment could improve the body's iron absorption. The study was conducted on primary school children (9-12 years) for 3 months. They were divided into two groups: those who consumed iron supplement as syrup (given twice a week), and those who consumed a synbiotic mixture-Lactobacillus plantarum Dad 13 and fructo-oligosaccharide in fermented milk (six times per week). As a result, the children did not present significant differences because they came from wealthy families and did not suffer from food shortage. However, a greater presence of E. coli bacteria was observed among those who drank only iron supplement syrup, and a high number of Bifidobacteria was counted in the stool of those who consumed the synbiotic formula with fermented milk [33].
Another article examined the in vitro effects of inulin on the availability of iron in two probiotic yogurts (milk-and soy-based yogurts). The evaluation was made before and after yogurts' incubation, monitoring the dialyzable iron fraction, the cell ferritin formation, and cell associated iron. The Caco-2 cell line is a line of human adenocarcinoma cells that is a useful model for studying iron absorption, used in the mentioned study. After incubating both yogurts with and without inulin, the formation of ferritin was higher. This demonstrates that inulin by itself does not directly influence iron absorption, but has a positive impact when used by the probiotic cells [110].
Nanoparticles, such as iron oxide nanoparticles, are a further alternative to dietary supplements. They simultaneously retain the organoleptic properties in food. An innovative formulation is composed of iron oxide nanoparticles, pectin, and lactic acid bacteria. These three components assembled could offer the best form of protection for the microorganisms and for the administration as well as insurance of the safe arrival of soluble iron to the intestine [101].
A different study examines the effects of synbiotic intake on patients with type 2 diabetes as many metabolic complications are associated with changes in serum minerals. The intake of synbiotics can have positive effects on nondiabetic patients and on type 2 diabetic patients, showing significant changes regarding serum iron absorption [111]. However, extensive studies need to confirm the probiotic, prebiotic, and synbiotic positive effect in iron deficiency treatment. Specific recommendations regarding strain, dosage, synbiotic iron formulations, and metabolic implications need to be clarified.

Conclusions
Iron metabolism involves many processes and, even excluding the genetic defects, there are several causes for iron deficiency in humans. Therefore, multiple approaches for increasing iron absorption exist. Probiotics, prebiotics, and synbiotic formulations can be employed to achieve the desired iron concentration. Nowadays, studies reveal multiple mechanisms through which these formulations can help in regulating the iron deficiencies. Understanding probiotics' capacity to act as carriers for iron, to convert unavailable iron into its available form, or to create metabolites that indirectly increase the iron absorption in the gut is essential in this approach. These formulations need to become the focus of further studies because current studies, although shortcoming in number, already have promising results. Gut microbiota modulation through pro-and prebiotic intake can affect the iron absorption, but the type of ingested iron is also essential. Cytotoxicity of the unabsorbed iron on the gut cells (enterocytes) needs to be extensively addressed and tailored foods can bring an outstanding approach in iron deficiency treatment.

Conflicts of Interest:
The authors declare no conflict of interest.