Iron Fortification of Lentil (Lens culinaris Medik.) to Address Iron Deficiency

Iron (Fe) deficiency is a major human health concern in areas of the world in which diets are often Fe deficient. In the current study, we aimed to identify appropriate methods and optimal dosage for Fe fortification of lentil (Lens culinaris Medik.) dal with FeSO4·7H2O (ferrous sulphate hepta-hydrate), NaFeEDTA (ethylenediaminetetraacetic acid iron (III) sodium salt) and FeSO4·H2O (ferrous sulphate mono-hydrate). We used a colorimetric method to determine the appearance of the dal fortified with fortificants at different Fe concentrations and under different storage conditions. Relative Fe bioavailability was assessed using an in vitro cell culture bioassay. We found that NaFeEDTA was the most suitable fortificant for red lentil dal, and at 1600 ppm, NaFeEDTA provides 13–14 mg of additional Fe per 100 g of dal. Lentil dal sprayed with fortificant solutions, followed by shaking and drying at 75 °C, performed best with respect to drying time and color change. Total Fe and phytic acid concentrations differed significantly between cooked unfortified and fortified lentil, ranging from 68.7 to 238.5 ppm and 7.2 to 8.0 mg g−1, respectively. The relative Fe bioavailability of cooked fortified lentil was increased by 32.2–36.6% compared to unfortified cooked lentil. We conclude that fortification of lentil dal is effective and could provide significant health benefits to dal-consuming populations vulnerable to Fe deficiency.


Introduction
Lentil (Lens culinaris Medikus) is an important legume crop, cultivated for food and feed since prehistoric times. As a source of dietary protein, lentil can be combined with cereals to prepare human diets and animal feeds that provide a balance of essential amino acids and essential micronutrients such as iron, zinc and selenium [1,2]. Lentil is a good source of non-heme iron, ranging from 73 to 90 mg kg −1 [3]. The crude protein content (N × 6.25) of Western Canadian lentil is reported to range from 25.8 to 27.1% [4]. Lentil also is considered to be a starchy legume as it contains 27.4-47.1% starch, with a significant level of amylose (23.5-32.2)% [5,6]. Although lentil is a good source of intrinsic Fe, the bioavailability/absorption is low [7]. These authors reported that the mean Fe absorption from lentil dal was 2.2%, which was significantly lower than the 23.6% observed for a similar amount of Fe given as ferrous sulphate to women with poor Fe status. Low bioavailability may be due to the presence of phytic acid and polyphenols in the lentil dal [7,8].
have potential for fortifying dehulled lentil seed [13]. The second objective was fulfilled by conducting studies to help standardize the protocol for lentil fortification. These included assessments of the appropriate dose of Fe solution, selection of the most appropriate fortification method in the context of changes in organoleptic properties and storage capability, assessment of the best temperature for drying lentil after the addition of fortificants, and the effect of fortification on boiling time.

Materials and Methods
The procedure followed for development of a lentil fortification protocol is shown in Figure 1, and is discussed below.
Nutrients 2017, 9,863 3 of 19 second objective was fulfilled by conducting studies to help standardize the protocol for lentil fortification. These included assessments of the appropriate dose of Fe solution, selection of the most appropriate fortification method in the context of changes in organoleptic properties and storage capability, assessment of the best temperature for drying lentil after the addition of fortificants, and the effect of fortification on boiling time.

Materials and Methods
The procedure followed for development of a lentil fortification protocol is shown in Figure 1, and is discussed below. Flow chart for development of a lentil fortification protocol. a Oven dried, soaked and oven dried (DSD); sprayed followed by shaking and drying (SSD); rinsed, oven dried, soaked, and oven dried (RDSD); directly soaked in Fe solution (SD) and rinsed, soaked, and oven dried (SRD).

Selection of Lentil Genotype and Dehulled Lentil Product Type
Fifteen red cotyledon lentil cultivars/genotypes were analyzed to estimate the concentration (ppm) of Fe in seeds (data not shown). One widely grown and popular cultivated red lentil cultivar, CDC (Crop Development Centre) Maxim, developed at the Crop Development Centre, University of Saskatchewan, Saskatoon, SK, Canada, was selected for fortification studies due to its having a high Fe concentration (75-90 ppm) compared to other red lentil cultivars grown in Saskatchewan [21].
Four different types of dehulled lentil products are usually available in the red lentil market: polished football (dehulled, unsplit), polished splits, unpolished football and unpolished splits Flow chart for development of a lentil fortification protocol. a Oven dried, soaked and oven dried (DSD); sprayed followed by shaking and drying (SSD); rinsed, oven dried, soaked, and oven dried (RDSD); directly soaked in Fe solution (SD) and rinsed, soaked, and oven dried (SRD).

Selection of Lentil Genotype and Dehulled Lentil Product Type
Fifteen red cotyledon lentil cultivars/genotypes were analyzed to estimate the concentration (ppm) of Fe in seeds (data not shown). One widely grown and popular cultivated red lentil cultivar, CDC (Crop Development Centre) Maxim, developed at the Crop Development Centre, University of Saskatchewan, Saskatoon, SK, Canada, was selected for fortification studies due to its having a high Fe concentration (75-90 ppm) compared to other red lentil cultivars grown in Saskatchewan [21].
Four different types of dehulled lentil products are usually available in the red lentil market: polished football (dehulled, unsplit), polished splits, unpolished football and unpolished splits (Figure 2a). The Fe concentration in each product type was measured to determine the range of variability in Fe concentration. The product types then were used in a fortification study and samples of 200 g of each product type were mixed with 20 mL of NaFeEDTA solution (1600 ppm Fe) with four replications. The best product type in relation to uniformity of absorption of Fe solution, drying time and concentration of Fe in the fortified product was selected. The statistical analysis was conducted using SAS version 9.4 (SAS Inc., Cary, NC, USA). One-way analysis of variance (ANOVA) was used to compare the Fe concentration of unfortified and fortified red lentil product types. The least significant difference (LSD) was calculated and the level of significance set at p < 0.05. ( Figure 2a). The Fe concentration in each product type was measured to determine the range of variability in Fe concentration. The product types then were used in a fortification study and samples of 200 g of each product type were mixed with 20 mL of NaFeEDTA solution (1600 ppm Fe) with four replications. The best product type in relation to uniformity of absorption of Fe solution, drying time and concentration of Fe in the fortified product was selected. The statistical analysis was conducted using SAS version 9.4 (SAS Inc., Cary, NC, USA). One-way analysis of variance (ANOVA) was used to compare the Fe concentration of unfortified and fortified red lentil product types. The least significant difference (LSD) was calculated and the level of significance set at p < 0.05.

Selection and Evaluation of the Most Suitable Fe Fortificant for Lentil
The selection of the most appropriate Fe fortificant is challenging due to possible interactions between the food product and the Fe compound. Three water-soluble Fe compounds, FeSO4·7H2O, NaFeEDTA and FeSO4·H2O were selected from a list of iron fortificants published in the WHO and

Selection and Evaluation of the Most Suitable Fe Fortificant for Lentil
The selection of the most appropriate Fe fortificant is challenging due to possible interactions between the food product and the Fe compound. Three water-soluble Fe compounds, FeSO 4 ·7H 2 O, NaFeEDTA and FeSO 4 ·H 2 O were selected from a list of iron fortificants published in the WHO and FAO document "Guidelines on Food Fortification with Micronutrients" [13]. The FeSO 4 ·7H 2 O and FeSO 4 ·H 2 O were supplied by Crown Technology, Inc., Indianapolis, IN, USA, and NaFeEDTA by Akzo Nobel Functional Chemicals, LLC, Chicago, IL, USA. The three fortificants were food grade and were selected on the basis of their relative bioavailability, interaction with the food vehicle and cost of fortification [14].

Techniques Used for Lentil Fortification
An experiment was designed to determine the most appropriate method for fortifying dehulled, polished, football lentil dal with an Fe solution prepared with FeSO 4 ·7H 2 O, one of the three Fe fortificants studied. Five methods were used to fortify lentil dal with FeSO 4 ·7H 2 O solution (1600 ppm Fe) at 10 mL fortificant solution/100g dal. The 1600 ppm Fe concentration was selected with the aim that this concentration may provide a major part of the recommended daily allowances (RDAs) for humans. However, each method to fortify lentil dal is described below.
Method 1 (Dry-Soak-Dry). Lentil dal was oven dried at 80 • C for 10 min, soaked in 10 mL of fortificant solution for 2 min, and then dried again at 80 • C to obtain a moisture content of 14%. Method 3 (Rinse-Dry-Soak-Dry). The third method consisted of rinsing 100 g dal samples under a continuous flow of deionized water for 30 s followed by oven drying at 80 • C for 10 min. The dried sample then was soaked in the fortificant solution (10 mL fortificant solution/100 g lentil) for 2 min and then placed in the oven again for 15 min at 80 • C to reduce the moisture level to 14%.
Method 4 (Soak-Dry). Lentil dal was soaked in fortificant solution followed by oven drying at 80 • C to 14% moisture.
Method 5 (Soak-Rinse-Dry). Lentil dal was soaked in fortificant solution and then rinsed with deionized water for 30 s, followed by oven drying at 80 • C to 14% moisture.

HunterLab Colorimetric Measurements of Fe-Fortified Lentil Samples
The color of the Fe-fortified lentil sample from each of the five fortification methods was measured using a HunterLab instrument (Hunter Associates Laboratory Inc., Reston, VA, USA) to allow comparison with unfortified control samples. For each method, four samples were assessed. The dimensions L*, a* and b* were compared with those of the control sample, where L* indicates lightness (ranging from 0 to 100), a* indicates red (+) and green (−) and b* indicates yellow (+) and blue (−) with a range of +80 to −80 [22]. The L*, a* and b* values were analyzed using ANOVA in SAS 9.4.

Assessment of Appropriate Temperature and Duration for Drying Fortified Lentil Dal
Electric heat lamps of three power levels (100, 200 and 250 watts) (Trileaf Distributor) were used to dry fortified football dal after spraying with fortificant solution. The distance between the bulb and the lentil dal surface was 15 cm. Samples of 100 g of dal were fortified with 10 mL of FeSO 4 ·7H 2 O solution (1600 ppm Fe concentration). The maximum temperature ( • C) in the middle of the fortified dal sample during drying with the three bulb types and shaking using a Barnstead Thermolyne M49235 Bigger Bill Orbital Shaker (Sigma-Aldrich Corp.) was assessed using a thermometer (VWR Scientific, Chicago, IL, USA). The time to achieve 14% moisture for each sample was recorded for each treatment method. Both temperature and drying time were assessed three times and the mean temperature and drying time were calculated.

Estimation of Fe Concentration in Fortified Lentil Dal Samples by Flame Atomic Absorption Spectrophotometry (F-AAS)
The iron concentration in the fortified lentil dal was analyzed by flame atomic absorption spectrophotometry (F-AAS, Nova 300, Analytic Jena AG, Konrad-Zuse-Strasse, Neu-Ulm, Germany). Each sample was sub-sampled and 0.5 g was digested in a 30-mL digestion tube with HNO 3 -H 2 O 2 using an automatic digester (Vulcan 84, Questron Technology, Ontario, CA, USA). All chemicals (nitric acid (70%), hydrogen peroxide (30%) and hydrochloric acid (37%)) used for digestion were of analytical grade. The digestion was repeated twice, with three technical replications per repeat. In the digestion chamber, a total of 72 samples were digested in each run, along with eight standards (yellow lentil laboratory check) and four blanks. Samples were first digested with HNO 3 at 90 • C for 45 min, followed by addition of 5 mL of 30% H 2 O 2 and then further digested for another 65 min. The solutions were then reduced with 3 mL of 6 M HCl, followed by heating at 90 • C for 5 min prior to cooling to room temperature. All sample solutions were then diluted with deionized water to a volume of 25 mL. Six mL of each of the digested samples was then used to determine the Fe concentration as described previously [23]. The Fe concentration values were analyzed using ANOVA in SAS 9.4 to determine differences for Fe concentration among the fortified lentil samples within each of the three fortificants at concentrations ranging from 100 to 3200 ppm. The LSD was calculated and the level of significance set at p < 0.001.

Assessment of the Appropriate Dose of Fe Solution
A total of 51 different solutions of the three fortificants (17 solutions of each fortificant with Fe concentrations of 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000 and 3200 ppm) were prepared to fortify dehulled lentil dal samples. Ten mL of each fortificant solution at each Fe concentration was added to a 100-g dal sample and processed using the SSD (Spray-Shake-Dry) method described earlier. Twenty-five Fe solutions were prepared using the three Fe fortificants at eight concentrations (200, 400, 800, 1200, 1600, 2000, 2800 and 3200 ppm of Fe plus deionized water as the control) to assess the effect of increasing fortificant concentration on the pH of the solutions, which was measured three times for each solution using a pH meter (Oakton H 2 O proof BNC pH tester, Cole-Parmer Scientific Experts, Montreal, QC, Canada). Data were analyzed using SAS 9.4.

HunterLab Colorimeter Measurements of Stored Fe-Fortified Dal Samples
The initial color of Fe-fortified lentil dal samples was measured using a HunterLab (Hunter Associates Laboratory Inc., Reston, VA, USA) instrument. Twenty-seven samples (nine concentrations of each of the three Fe fortificants) and one control (unfortified lentil dal) with four replications were scored for their L*, a* and b* values. Samples of each treatment were stored individually at room temperature (25 • C) for one year in clear plastic bags (Ronco, Toronto, ON, Canada), similar to methods traditionally used to store dal products. After six months and one year of storage, the L*, a* and b* values of the lentil dal again were measured to determine if any color change had occurred. The one-year storage period was considered an approximate maximum storage period from processing to consumption by dal consumers. The L*, a* and b* values were analyzed using ANOVA in SAS 9.4.

Boiling Time Estimation of Fortified Lentil Dal Samples
Three fortified dal samples (FeSO 4 ·7H 2 0, NaFeEDTA and FeSO 4 ·H 2 O at 1600 ppm Fe concentration) and one unfortified control were used to determine if differences existed in boiling time between fortified samples and the control. Two hundred fifty grams of each of the lentil dal samples were cooked in 1L of deionized water containing 5 g of NaCl on a single burner gas stove at 104 • C. The boiling time was recorded as the point when >90% of the dehulled lentils were softened to the point that the mixture with water produced a thickened soup, a method of preparation like that commonly used in the South Asian Region [24]. This study was replicated three times and data were analyzed using SAS 9.4.

Relative Fe Bioavailability and Phytic Acid Content of Fortified Lentils
Lentil dishes were prepared for four different samples, including Fe-fortified lentil and the control (unfortified lentil). Both fortified and control samples were rinsed with 18 MΩ deionized water. A traditional Bangladeshi lentil dish (dal) was prepared in stainless steel cookware using a traditional Bangladeshi recipe [24] where salt, turmeric powder, onion, canola oil and deionized water were used as ingredients at a 15:75:5:3:2 ratio. The prepared dish was cooled to room temperature for 2 h, frozen at −80 • C for 24 h, freeze dried using a FreeZone 12 Liter Console Freeze Dry System with Stoppering Trays (Labconco, model 7759040, Kansas City, MO, USA) for 72 hand stored at room temperature [25]. Ten grams of freeze-dried dal from each dish was finely grounded and sent to the USDA-ARS Robert Holley Center for Agriculture and Health (Ithaca, New York, NY, USA) to assess iron concentration and bioavailability using an in vitro digestion/Caco-2 cell culture bioassay [26]. Total Fe concentration from the cooked lentil samples was measured using a standard HNO 3 -HClO 4 method and atomic absorption spectrophotometry [23]. The phytic acid (total phosphorus) test kit (Megazyme International, County Wicklow, Ireland), a simple, quantitative, colorimetric and high throughput method [25,27], was used for the measurement and analysis of phytic acid in the four cooked lentil samples used for the bioavailability assessment. The ANOVA was conducted using SAS 9.4 to determine differences in iron concentration, relative iron bioavailability and phytic acid concentration among the cooked fortified lentil dishes. The LSD was calculated and the level of significance set at p < 0.001.

Selection of Dehulled Lentil Product Type for Fortification
Prior to fortification, no significant differences in Fe concentration existed among product types (70-73 ppm Fe) (Figure 2b). After fortification with 200 ppm of Fe, significant differences in Fe concentration were observed among product types (Figure 2c). The highest Fe concentrations were observed in fortified unpolished split (196.7 ppm) and polished football (191.5 ppm) dal. Polished football dal, which is typically polished with water and/or vegetable oil after milling, performed best in the context of uniformity of mixing with the fortificant solution and drying in the shaker-when placed in the shaker, the polished football dal moved more and agitated more quickly in the mixing trays. This helped to distribute the heat over the surface of the dal, hence it dried more uniformly and did not stick to the tray surface when wet. Selection of dehulled lentil rather than whole lentil was important, because removal of the seed coat has a significant effect on reducing the levels of polyphenolic compounds, thereby increasing Fe bioavailability [21].
For commercial-scale fortification, any of the four lentil product types potentially could be fortified. Consumer demand and the relative cost and availability of the various processing techniques would be important considerations. Successful fortification to produce fortified food depends on the interactions among the food vehicle, fortificant and the fortification technique. Dehulled lentil dal is available in three colors-red, yellow and green. Red cotyledon lentil was selected for fortification since it is the most widely consumed form of lentil dal, with wide acceptability in South Asia and the Middle East [28]. Consumers from some countries in these regions consume lentil as an essential component of their typical daily diet. Yellow and green lentil dal samples also were fortified and no significant differences were observed for final Fe concentration when fortified with similar concentrations of Fe fortificants (data not shown). Hence, any of red, yellow or green lentil dal could be fortified with the Fe fortificants.

Selection and Evaluation of the Most Suitable Fe Fortificant for Lentil
The success of food fortification programs is based on the chemistry between food vehicles and the fortificant selected to fortify foods [29]. Different food vehicles may contain different moisture levels and oxidizing agents that can react with fortificants and develop rancidity, metallic taste, off-color or degradation of vitamins, all factors that can influence bioavailability [30,31].
NaFeEDTA was shown previously to be two to four times more effective for increasing absorption of dietary Fe in humans compared to FeSO 4 and ferrous fumarate [32]. It also was reported that Fe absorption was increased by using a mixture of FeSO 4 and NaFeEDTA, instead of NaFeEDTA alone [33]. In another study, NaFeEDTA was proven to be a promising cost effective, water-soluble and highly bioavailable Fe fortificant that improved the Fe status of Vietnamese woman when consumed for 6 months (10 mg Fe for 6 days/week) [33]. These authors also reported that the prevalence of Fe deficiency and Fe deficiency anemia were reduced from 62.5% to 32.8% and from 58.3% to 20.3%, respectively.
The effect of NaFeEDTA-fortified wheat flour on urinary zinc extraction was studied and no effect was found in children [34]. Another study revealed no significant negative effects of NaFeEDTA-fortified bread (bread made with 100 g of NaFeEDTA-fortified wheat flour that contained 5 mg of Fe and was consumed as a single meal per day) consumption on Zn and Ca metabolism, and that NaFeEDTA might increase Zn absorption and Fe bioavailability from the low bioavailability diets [35]. In another study, NaFeEDTA was shown to have no influence on absorption or urinary excretion of Mn [36]. NaFeEDTA-fortified fish sauces also increased significantly the amounts of Hb and serum ferritin when provided to iron-deficient, anemic school children in Cambodia [37].
The review of the safety and efficacy of different dietary strategies for improving Fe status revealed that there are no reported data that demonstrate specific adverse effects of iron-fortified food items [38]. Moreover, the daily dose of Fe is much lower from fortified food than on supplementation [39]. The joint FAO/WHO Expert Committee on Food Additives (JECFA) summarized data on the basis of acute and chronic toxicity, reproduction, carcinogenicity, genotoxicity and teratogenicity of EDTA and its salts, such as NaFeEDTA [40]. The Committee also evaluated biochemical and toxicological aspects of using NaFeEDTA as a fortificant and stated that: (i) Fe from NaFeEDTA is released from the chelate to the common non-heme iron pool before Fe absorption; (ii) a very small fraction (1-2%) of NaFeEDTA is absorbed intact and is rapidly and completely excreted via the kidneys in the urine; (iii) dietary Fe fortification with NaFeEDTA does not increase the risk of iron accumulation in iron-replete individuals, and has no negative influence on the absorption of other micronutrients, such as Zn; and (iv) NaFeEDTA has low oral toxicity and does not induce gene mutations when tested with bacterial and mammalian cells in vitro. In addition, considering the cost of fortificant, NaFeEDTA is more expensive compared to FeSO 4 ·7H 2 O and FeSO 4 ·H 2 O, but its extra cost can be offset by its higher bioavailability in phytate-rich foods such as lentil [14].

Techniques Used for Lentil Fortification
Significant variation in Fe concentration was found among the five methods used to fortify lentil dal. The highest concentrations of Fe were found with the DSD (lentil dal oven dried, soaked, followed by oven drying) and SSD (lentil dal sprayed with fortificant solution followed by shaking and drying) methods ( Figure 3). Although the highest Fe absorption into the lentil seed was observed with DSD, the discoloration (increased darkness) of the final product may cause concern in the context of expected consumer preferences and longer fortification time (Figure 4). The homogeneity of Fe concentration was tested by randomly selecting six samples from the mixing tray. All samples contained similar amounts of Fe (215-220 ppm) after fortification.

HunterLab Colorimetric Measurements of Fe-Fortified Lentil Samples
The HunterLab results indicated significant variation for all three scales (L*, a* and b*), indicating off-color development due to fortification (Figure 4b-1-b-3). The highest values for all three scales were found for the unfortified control lentil dal sample. The lowest L* value was found for the DSD sample, whereas the lowest a* and b* values were found for the samples produced by the SD, RDSD and DSD methods. The L*, a* and b* values ranged from 46.3 to 52.8, 25.3 to 33.1 and 36.6 to 44.6, respectively. The shortest processing time was required with the SSD method (Figure 4b-4), which also generated off-color but significantly less compared to the SD, RDSD and DSD methods.

Assessment of Appropriate Temperature and Duration for Drying Fortified Lentil Dal
Temperature has been shown to have a significant effect on the drying time required to achieve a level of moisture suitable for safe storage [41]. The results from the assessment of appropriate temperature and duration for drying fortified lentil dal showed that with an increase in temperature caused by raising the light bulb wattage, there was an increase in the temperature ( • C) of both the aluminum foil tray used for fortification and the fortified lentil seed. An inverse relationship was observed between total drying time and temperature ( Figure S1). The temperature used to dry fortified lentil dal should be optimized to avoid off-color development, as a relationship between temperature and off-color development in fortified foods has been observed [30]. Using the 250-watt bulb, the temperature rose to 75 • C, which dried the fortified lentil dal in the shortest time (12-14 min). The moisture content of the fortified dal was approximately 14%, which is similar to the moisture content (%) of dehulled lentil dal (13)(14)% that is commercially available in the local market [42]. During fortification, lentil dal was treated with fortificant solution and then heat was applied to dry the product. This process might reduce the level of phytate and phenolics level to some extent, and enhance the bioavailability of both Fe and Zn [43].  (Figure 4b-4), which also generated off-color but significantly less compared to the SD, RDSD and DSD methods.

Assessment of Appropriate Temperature and Duration for Drying Fortified Lentil Dal
Temperature has been shown to have a significant effect on the drying time required to achieve a level of moisture suitable for safe storage [41]. The results from the assessment of appropriate temperature and duration for drying fortified lentil dal showed that with an increase in temperature caused by raising the light bulb wattage, there was an increase in the temperature (°C) of both the aluminum foil tray used for fortification and the fortified lentil seed. An inverse relationship was observed between total drying time and temperature ( Figure S1). The temperature used to dry fortified lentil dal should be optimized to avoid off-color development, as a relationship between temperature and off-color development in fortified foods has been observed [30]. Using the 250-watt bulb, the temperature rose to 75 °C, which dried the fortified lentil dal in the shortest time (12-14 min). The moisture content of the fortified dal was approximately 14%, which is similar to the moisture content (%) of dehulled lentil dal (13)(14)% that is commercially available in the local market [42]. During fortification, lentil dal was treated with fortificant solution and then heat was applied to dry the product. This process might reduce the level of phytate and phenolics level to some extent, and enhance the bioavailability of both Fe and Zn [43].

Assessment of the pH of Solutions Prepared with Three Fe Fortificants over a Range of Concentrations
Measurement of pH over a range of concentrations of the Fe fortificants showed an inverse relationship between pH and an increase in the concentration of Fe in the solution. The pHs of the three fortificant solutions were lower (<5) than that of deionized water (6.7). The rate of decrease of pH with an increase in Fe concentration was highest for FeSO4·H2O, followed by FeSO4·7H2O and NaFeEDTA ( Figure 5). The pH of the fortificant solution would have an effect on the solubility of Fe [44]. Both pH and redox potential influence the oxidation state of Fe, and both the Fe +2 and the Fe +3 form are used for fortification. Both have unfilled orbits that can react with electron-rich components, thus influencing organoleptic attributes and bioavailability [45]. The oxidation-reduction reactions (redox potential) in fortified foods, due to the addition of Fe that can react with phenolic compounds, cause off-color development [43]. Ferrous ion oxidizes to the ferric form as redox potential increases, but remains constant at a lower redox potential [30,44]. The solubility of FeSO4 in 0.1 M HCI was

Assessment of the pH of Solutions Prepared with Three Fe Fortificants over a Range of Concentrations
Measurement of pH over a range of concentrations of the Fe fortificants showed an inverse relationship between pH and an increase in the concentration of Fe in the solution. The pHs of the three fortificant solutions were lower (<5) than that of deionized water (6.7). The rate of decrease of pH with an increase in Fe concentration was highest for FeSO 4 ·H 2 O, followed by FeSO 4 ·7H 2 O and NaFeEDTA ( Figure 5). The pH of the fortificant solution would have an effect on the solubility of Fe [44]. Both pH and redox potential influence the oxidation state of Fe, and both the Fe +2 and the Fe +3 form are used for fortification. Both have unfilled orbits that can react with electron-rich components, thus influencing organoleptic attributes and bioavailability [45]. The oxidation-reduction reactions (redox potential) in fortified foods, due to the addition of Fe that can react with phenolic compounds, cause off-color development [43]. Ferrous ion oxidizes to the ferric form as redox potential increases, but remains constant at a lower redox potential [30,44]. The solubility of FeSO 4 in 0.1 M HCI was reported to decrease by 74% with changes in pH over the range of 2-6, but remained constant for NaFeEDTA [45]. In this study, an increase in FeSO 4 concentration resulted in a faster rate of pH reduction in comparison to NaFeEDTA. Moreover, to obtain a similar amount of soluble Fe at a specific pH, more FeSO 4 is required than NaFeEDTA. This may cause a major change in the organoleptic characteristics of lentil dal. This study showed that NaFeEDTA would be a better choice than FeSO 4 for fortification of lentil dal.
Nutrients 2017, 9, 863 11 of 19 reported to decrease by 74% with changes in pH over the range of 2-6, but remained constant for NaFeEDTA [45]. In this study, an increase in FeSO4 concentration resulted in a faster rate of pH reduction in comparison to NaFeEDTA. Moreover, to obtain a similar amount of soluble Fe at a specific pH, more FeSO4 is required than NaFeEDTA. This may cause a major change in the organoleptic characteristics of lentil dal. This study showed that NaFeEDTA would be a better choice than FeSO4 for fortification of lentil dal.

Estimation of Fe Concentration in Fortified Lentil Dal Samples using F-AAS
The concentration of Fe in fortified lentil dal increased with an increase in Fe concentration in the fortificant solution (Table 1). Off-color development also increased gradually with an increase in the Fe concentration of the fortificant (Table 2). Table 1. Fe concentration (ppm) in polished football lentil dal samples prepared using three fortificants (FeSO4·7H2O, NaFeEDTA and FeSO4·H2O) at concentrations ranging from 100-3200 ppm.

Estimation of Fe Concentration in Fortified Lentil Dal Samples Using F-AAS
The concentration of Fe in fortified lentil dal increased with an increase in Fe concentration in the fortificant solution (Table 1). Off-color development also increased gradually with an increase in the Fe concentration of the fortificant (Table 2). 349.0 ± 1.8 g 326 ± 3.1 h 381.7 ± 3.6 j a Mean ± SD. Mean scores for Fe concentration followed by different letters within columns are significantly different (p < 0.001). Table 2. Lightness (L*), redness (a*) and yellowness (b*) scores of fortified lentil samples prepared using FeSO 4 ·7H 2 O, NaFeEDTA and FeSO 4 ·H 2 O at concentrations ranging from 100 to 3200 ppm after six months and after one year of storage.

Assessment of the Appropriate Dose of Fe
Consideration of the appropriate dose of Fe is important for optimizing the amount of fortificant required to provide a major part of the estimated average requirement (EAR) for available Fe. The WHO has suggested suitable iron compounds to fortify specific food vehicles [13]. For instance, NaFeEDTA was suggested to fortify high extraction wheat flour, sugar, soy sauce, and fish at different rates. The bioavailability of Fe depends on the levels of various compounds present in the food vehicle, e.g., phytate, dietary fiber, tannins and other polyphenols [25,46]. These components can reduce the absorption of micronutrients, e.g., Fe, Zn. Moreover, Fe of plant origin is exclusively non-heme Fe, which is less bioavailable than the heme Fe from animal sources [46,47]. In this study, lentil dal fortified with three different fortificants showed an increase in Fe concentration with an increase in the Fe concentration in the fortificant solution. Lentil seed may exhibit a wide range in Fe concentration [7]. According to the FAO and WHO, EARs for iron having 10% bioavailability are 29.4 and 10.8 mg Fe day −1 for females and males, 19-50 years of age, respectively [13]. Therefore, 50 g of unfortified dehulled lentil could provide approximately 3.5 mg of Fe, based on the Fe concentration in the control lentil dal sample. The bioavailability may decrease if the dal is prepared with spices or condiments and is eaten with other foods such as rice, bread or vegetables, which may contain phytate, polyphenols or other components that reduce the absorption of Fe. To obtain a major portion of daily Fe from food fortificants, an optimum dose should be recommended. In this study, it was shown that lentil dal fortified with 1600 ppm of Fe could provide approximately 130-140 ppm of Fe per 100 g of lentil. Therefore, 50 g of fortified lentil could provide approximately 10 mg of Fe (6.5-7 mg of Fe from the fortificant + 3.5 mg from the lentil). This could provide a major portion of the EAR. Currently, 30-45 mg kg −1 ferrous sulphate and 250 mg kg −1 NaFeEDTA are used to fortify wheat flour and soy/fish sauce, respectively [13].

HunterLab Colorimeter Measurements of Stored Fe-Fortified Dal Samples
Color attributes influence the acceptability of a food product to consumers. The L*, a* and b* scores were significantly decreased with an increase in Fe concentration provided by any of the fortificants. Significant variation in color was observed among lentil dal samples fortified with the three fortificants at any concentration. Samples fortified with NaFeEDTA had higher L*, a* and b* scores, similar to those of the control, indicating less off-color development when compared to dal samples fortified with FeSO 4 ·7H 2 O or FeSO 4 ·H 2 O ( Figure 6).
The usual expectation for any Fe-fortified food product is that it does not exhibit any off-color. The dark color of the micropylar area of fortified lentil dal possibly could be used as an indicator to help consumers distinguish between fortified and unfortified lentil dal, where the micropylar region is white. The L*, a* and b* color values for the fortified lentil dal samples showed some inverse relationships with the progress of storage time (Table 2). Lightness (L*) increased slightly, but a* and b* decreased in all of the fortified lentil dal samples over time. Initially, just after fortification, the L* value ranged from 50.6 (unfortified control) to 42.2 (fortified with 3200 ppm of FeSO 4 ·7H 2 O), which was similar to the samples fortified with FeSO 4 ·H 2 O (42.6). The range was narrower for the L* value of samples fortified with NaFeEDTA (50.6 to 46.4) ( Table 2). For all three fortificants, after 6 months and one year of storage of fortified lentil dal, there was an increasing trend in L*, but a decreasing trend for the a* and b* values ( Table 2). The non-significant differences in the L*, a* and b* scores for the unfortified and fortified lentil samples provides assurance that the minor changes observed will not influence consumer acceptability. The L*, a* and b* values for fortified lentil dal, prepared with the three fortificants at 1600 ppm of Fe, showed numerical decreases, but these were not significant for the three storage periods, except for the L* and b* scores for the FeSO 4 ·7H 2 O-fortified and the NaFeEDTA-fortified samples, respectively (Figure 7). These small changes may be caused by the presence of very small amounts of lipid (1.52-2.95%) [48] that could increase the likelihood of lipid oxidation and result in off-color development over time.

Boiling Time Estimation of Fortified Samples Compared to the Unfortified Control
The boiling time of lentil dal is important and may influence consumer acceptability due to energy and time consumption during cooking. Compared to unfortified lentil dal, the fortified lentil dal should take equal or less time to cook, and have similar texture, taste and appearance after cooking. Among the four samples that were cooked to determine the variability in boiling time, all had similar cooking times ( Figure S2). Fortification had no significant influence on the boiling time of FeSO4·7H2O-, FeSO4·H2O-or NaFeEDTA-fortified samples compared to the control.

Iron Concentration, Relative Fe Bioavailability and Phytic Acid Concentration of Fortified Lentils
Significant differences were observed among fortified and unfortified lentil samples in Fe concentration, relative Fe bioavailability and phytic acid concentration (Table 3). Similar iron and

Boiling Time Estimation of Fortified Samples Compared to the Unfortified Control
The boiling time of lentil dal is important and may influence consumer acceptability due to energy and time consumption during cooking. Compared to unfortified lentil dal, the fortified lentil dal should take equal or less time to cook, and have similar texture, taste and appearance after cooking. Among the four samples that were cooked to determine the variability in boiling time, all had similar cooking times ( Figure S2). Fortification had no significant influence on the boiling time of FeSO 4 ·7H 2 O-, FeSO 4 ·H 2 O-or NaFeEDTA-fortified samples compared to the control.

Iron Concentration, Relative Fe Bioavailability and Phytic Acid Concentration of Fortified Lentils
Significant differences were observed among fortified and unfortified lentil samples in Fe concentration, relative Fe bioavailability and phytic acid concentration (Table 3). Similar iron and phytic acid concentrations were observed in FeSO 4 ·7H 2 O-and NaFeEDTA-fortified samples. The unfortified lentil samples were statistically different than the three fortified samples for all four measurements. The relative bioavailability was similar for all three fortified lentil dal samples. Iron concentration and relative Fe bioavailability ranged from 68.7 to 238.5 ppm and 68.3 to 104.9, respectively. The relative Fe bioavailability of the three cooked fortified lentil dal samples was 1.4 to 1.5 times higher than that of unfortified cooked lentil sample (control). Phytic acid concentration ranged from 7.2 to 8.0 mg g −1 . Table 3. Mean iron (Fe) concentration (ppm), relative bioavailability (ng ferritin (mg protein) −1 ) and phytic acid concentration (mg g −1 ) of four cooked freeze-dried lentil samples. Fortification of lentil dal is more complex than fortifying flour, beverages and most other food products due to the requirement to apply fortificant solution to the surface of the dal. Considering all of the results from the various experiments, it was concluded that lentil dal could be used as a vehicle for Fe fortification and that NaFeEDTA was the most suitable Fe fortificant for lentil dal. These results represent baseline data for the commercial production of Fe-fortified lentil dal. This research is unique in the context of lentil dal fortification, and will be followed by sensory evaluation to select the most appropriate fortificant after evaluation of overall acceptability. Results from sensory evaluation with both uncooked and cooked fortified lentil dal compared favorably with the control and will be described in a subsequent manuscript. Community-based efficacy and effectiveness studies with fortified lentil in the target populations will be required. The bioavailability of fortified lentil in a large-scale human trial also could be evaluated to obtain an empirical estimate of the amount of Fe required to provide a major portion of the EARs for Fe in regions where Fe deficiency exists.

Cooked Lentil
Supplementary Materials: The following are available online at www.mdpi.com/2072-6643/9/8/863/s1, Figure S1: Effect of increasing light bulb wattage on temperature ( • C) and drying time (min) of fortified lentil samples, Figure S2: Effect of the three fortificants solution used to prepare three fortified lentil samples (FeSO 4 ·7H 2 O, NaFeEDTA and FeSO 4 ·H 2 O) on boiling time compared with one unfortified control sample. Different letters within each figure are significantly different (p < 0.05).