Food fortification is a considerable sustainable long-termstrategy for the prevention of micronutrient deficiencies in the generalpopulation. National Institute of Nutrition developed and tested the doublefortified salt (DFS) enriched in iron and iodine over the years. However, the bioavailabilityof iron supplied through DFS still remains to be studied.

In the current study,we assessed iron content and bioavailability from 12 different typical Indianrecipes prepared with either iodized salt (IS) or DFS using simulated in vitro digestion/Caco-2 cell model. Bothtotal and percent (%) dialyzable ironremained higher in DFS compared to IS based recipes. The dialyzable iron levelscorrelated with that of total iron only in IS but not in DFS based recipes,implying interaction with food matrix. The percent(%) dialyzability was higher from DFSbased recipes compared to IS. Further, the dialyzabilityfrom rice was higher compared to wheat- based DFS recipes.

Further, ferritininduction in Caco-2 cells, surrogate of iron absorption, significantlycorrelated with iron dialyzability fromDFS based recipes. The sodium hexametaphosphate (SHMP), a stabilizer in DFSformulation, did not influence the ferrous iron bioavailability but improvedthe solubility and bioavailability of ferric iron in Caco-2 cells. Together, theseresults suggest that use of DFS improves the total and bioavailable ironcompared to IS based recipes, and thus substantiate the previous efficacystudies in human subjects. Further, these results also indicate the enhancingeffect of SHMP on iron absorption in intestinal cells. Keywords: Doublefortified salt, iron, bioavailability, Caco-2 cells, in vitro digestion, and recipe    Introduction:Irondeficiency anemia is one of the leading nutritional deficiencies, affectingchildren and pregnant women in India (NFHS-4, 2015-16).

Iron-deficiency anemia,characterized by lower hemoglobin content (<12g/dL), is associated withhigher risk of morbidity and mortality in vulnerable segments of the population(Allen et al., 2000;Kotecha et al., 2011). The major etiologicalfactor for the widespread prevalence ofanemia in India may be attributed to the poor density and bioavailability ofiron from non- heme foods (Nairet al., 2009).

The phytic acid and polyphenols, abundant in plant foods,limits the absorption of iron (Lonnerdol et al., 2000; Hurrel 1997; Rao &Prabhavathi, 1983). Therefore, fortification of foods with iron isconsidered to prevent anemia and other micronutrient deficiencies in the generalpopulation (WHO report2006). Wheat and rice fortification demonstrated to improve iron statusin Indian children (Hurrelet al., 2002, Moretti et al., 2006; Radhika et al., 2011). However, the lackof centralized processing systems and household consumption patterns limits theapplication of above vehicles for fortification at this time.

  Common salt has several unique advantages, dueto its low cost, ubiquitous use by all sections of the population, particularlythe rural masses who are in fact at need. The successful introduction ofmandatory fortification of salt with iodine, and its impact on reducing goiter prevalence indeed support its utility asan ideal vehicle for fortification in developing countries (Sivakumar et al., 2002).Therefore, fortification of salt with iron and iodine is considered a potentialapproach to prevent iron and iodine deficiencies (Narsinga Rao et al., 1975, 1994; MicronutrientInitiative, Canada 1999).

The National Institute of Nutrition, ICMR, Indiahas developed and tested the double fortification of salt with ferrous sulfate and iodine (NIN-DFS), with sodiumhexametaphosphate (SHMP) as a stabilizer (Narsinga Rao et al., 1994; Sivakumar et al, 2002).Other DFS formulations developed by Micronutrient Initiative (1999, MI, Canada) wasbased on microencapsulation of iodine and using poorly soluble iron salt(ferrous fumarate), to prevent iodine loss. Extensive studieson DFS developed by NIN consistently demonstrated its acceptability and safety (Narasinga Rao et al., 1994,Sivakumar et al., 2001, Sivakumar et al.

, 2002; Nair et al., 1998; Brahamam etal., 1994; Nair et al., 1996). Further, DFS consumption has been shownto improve the haemoglobin levels incommunity settings, after correcting for confounding variables (Brahamam et al., 1994; Sivakumaret al., 2002).

In adult men, the meanabsorption of iron from the double fortified salt (with SHMP) was higher (6.1%)compared to ferrous sulphate alone (3.9%)when consumed with a rice-based meal (Narsinga Rao et al., 1994). However, the bioavailability of iron from typicalIndian recipes prepared with DFS needs to be assessed, particularly in thecontext of high phytic acid content, a potent inhibitor of iron absorption (Hurrel et al.,1997).

Early studies in humans and animals indicated anegative effect of inorganic-phosphate compounds on iron absorption (Greget et al., 1982). In contrast, polyphosphate compounds (i.e.

pyrophosphate) reported increasing solubility ofotherwise poorly soluble ferric iron compounds at neutral pH (Tian et al., 2016).Further, the addition of pyrophosphate to iron-fortifiedbouillon cubes improved the iron absorption in iron deficient women (Cercamondi et al., 2016).Interestingly, SHMP, the stabilizer used in DFS is also a polyphosphatecompound, but its effect on iron bioavailability was not yet studied. Here in we investigated the ironcontent and bioavailability of IS and DFS based typical recipes commonlyconsumed in India using simulated invitro digestion/Caco-2 cell model. To substantiate the results we have alsoinvestigated the effect of SHMP on solubility and intestinal absorption offerrous and ferric forms of iron.

 Materialsand Methods:Materials: Caco-2cells were obtained from National Centre for Cell Sciences, Pune, India. Allthe reagents and digestive enzymes were procured from Sigma Chemical Co,Bangalore, India unless otherwise specified. The double fortified and iodized saltswere procured from local companies.

Methods:Computationof iron and salt intakes in India: The average dietary intake of iron and saltin India are adopted from the National Nutrition Monitoring Bureau survey inrural population (NNMBreport 2012). Preparationof recipes: The list of ingredients and method of preparation of recipes withIS or DFS are exactly similar, and the details are provided in the supplementarydata file (Supplementary data file#1). The recipes were homogenized in akitchen blender to a fine homogenate and lyophilized to dryness and stored at-20°C, until further use.Estimation of dialyzable iron: The dialyzableiron content was estimated in a 6-well culture plate as described previously (Argyri et al., 2009).

Thelyophilized food homogenate (0.625g) were hydrated in saline (0.15 mmol/ LNaCl) for 30 min.  The pH of samples was adjustedto 2.8 with 6 mol/L HCl followed by an addition of 0.1 mL pepsin (40mg/mL).

Thefinal volumes were made to 12.5 mL with saline. Two mL aliquots weretransferred to six-well plates in triplicate and incubated for 2 h on a shaker.  At the end of this incubation, a transwellinsert fitted with a dialysis membrane was housed in individual wells of 6-wellplate, thus creating an apical and basolateral chamber.

The apical chamber was thenfilled with 2 mL PIPES buffer, pH 6.5. After 30 min, 0.5 mL of a pancreatin(2mg/mL)-bile salt mixture (12mg/mL) was added to the basal chamber contents, andthe incubation continued for another 2 h. At the end of incubation, the dialysatein the apical chamber was removed, and iron content was estimated by atomicabsorption spectrometry. Bioavailability in Caco-2 cells: Simulated in vitro digestion followed by ferritin induction in Caco-2 cells has been anextensively validated in vitro modelto study the iron absorption in Caco-2 cells (Glahn et al., 1998, Bejjani et al., 2007, Angela et al.

,2000). The description of the methodology is provided below.Caco-2cell culture:Caco-2 cells were obtained from the American Type Culture Collection (Rockville,MD), and grown as described previously (Palika et al., 2013). Briefly, cells, betweenpassages 28-35, were seeded at a density of 50,000 cells/cm2 in6-well plates and cultured in complete DMEM with 10% fetal bovine serum (FBS),supplemented with 1% NEAA, 0.

4 mM glutamine and 1% antibiotic-antimycotic solution and maintained at 37°C in anincubator with a 5% CO2/95% air atmosphere at constant humidity. Thecells were used 13-14 days post