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

Abstract

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.

Full text

nutrients
Article
Iron Fortification of Lentil (Lens culinaris Medik.) to
Address Iron Deficiency
Rajib Podder 1, Bunyamin Tar’an 1, Robert T. Tyler 2, Carol J. Henry 3, Diane M. DellaValle 4ID
and Albert Vandenberg 1, *
1Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada;
rap039@mail.usask.ca (R.P.); bunyamin.taran@usask.ca (B.T.)
2Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8,
Canada; bob.tyler@usask.ca
3College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5C9, Canada;
carol.henry@usask.ca
4Department of Nutrition and Dietetics, Marywood University, 2300, Adams Avenue, Scranton, PA 18509,
USA; ddellavalle@maryu.marywood.edu
*Correspondence: bert.vandenberg@usask.ca; Tel.: +1-306-221-2039
Received: 28 June 2017; Accepted: 7 August 2017; Published: 11 August 2017
Abstract:
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 FeSO
4·
7H
2
O (ferrous
sulphate hepta-hydrate), NaFeEDTA (ethylenediaminetetraacetic acid iron (III) sodium salt) and
FeSO
4·
H
2
O (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.
Keywords: lentil; iron; fortification; NaFeEDTA; FeSO4·7H2O; FeSO4·H2O
1. 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].
Nutrients 2017,9, 863; doi:10.3390/nu9080863 www.mdpi.com/journal/nutrients

Nutrients 2017,9, 863 2 of 19
Iron (Fe) is the most abundant element in the earth
0
s crust and is an essential micronutrient for
both plants and animals. In plants, Fe deficiency affects key metabolic processes such as the electron
transfer system for photosynthesis and respiration [
9
]. Iron deficiency in humans refers to a condition
in which an insufficient amount of bioavailable Fe results in Fe deficiency anemia [
10
]. This deficiency
has become a major nutritional disorder, widespread in both developing and developed countries [
11
].
The major consequences of Fe deficiency are reduction of physical activity, fitness and work capability,
a reduced ability to maintain body temperature, a lowered resistance to infection, and an increase in
mortality during pregnancy and in newborns [
12
]. According to Food and Agriculture Organization
(FAO) and World Health Organization (WHO) recommendations, the estimated daily average Fe
requirements for females and males 19–50 years of age are 29.4 mg and 10.8 mg, respectively, based on
10% bioavailability [13].
Several strategies are used around the world to address micronutrient malnutrition. Micronutrient
supplementation, dietary diversification, biofortification, food fortification, nutrition education, public
health interventions and food safety measures are approaches that can solely or in combination be
applied to address micronutrient deficiency in a target population [
14
]. Supplementation is an effective
means of providing immediate benefits to “at risk groups” but not for other household or community
members [
15
] since it requires supplemental Fe consumption on a long-term basis, in tablet form
for example. Dietary improvement through supplementation requires a change in dietary behavior,
and this process also requires changes in food supply and availability that may require a long time to
achieve success [
14
]. Also, public health intervention can help prevent micronutrient malnutrition,
but micronutrient malnutrition can also be associated with a high prevalence of microbial infection
that causes a variety of different diseases. Food fortification can overcome this limitation due to its
sustainability in improving the dietary quality of a targeted group or population without changing
dietary habits. Food fortification is a potentially cost-effective way to add micronutrients to processed
foods in a way that can rapidly mitigate micronutrient malnutrition [13].
A successful Fe fortification program was first reported in Canada in 1944, when the government
began fortifying wheat flour with Fe along with thiamine, riboflavin and niacin [
14
]. A remarkable
reduction in child mortality was observed from 102/1000 live births in 1944 (first year) to 61/1000
in 1947 in Canada [
16
]. During the twentieth century, Fe fortification became mandatory in several
developing countries, including Bolivia, Chile, Colombia, Costa Rica, Ecuador, Guatemala, Indonesia
and others [
17
]. In every country, either wheat or maize flour was chosen as the food vehicle.
The requirements for selecting an appropriate food vehicle for fortification were established by FAO in
1995 [
17
]. In 1980, the FDA (U.S. Food and Drug Administration) established a “Food Fortification
Policy” that was guided by six basic principles [
18
]. The WHO has recommended Fe compounds and
concentration for fortification of wheat flour in 13 countries [
19
]. To optimize iron bioavailability and
maintain the organoleptic attributes that influence consumer acceptability of fortified foods, selected
food vehicles and Fe fortificants need to be well matched. The food vehicle should be safe, widely
accepted by the target consumers, have good storage capability after fortification, and the added Fe
should be stable with high bioavailability [20].
Fortifying lentil with suitable Fe fortificants is a research area with potential application to reduce
Fe deficiency. We hypothesized that it would be possible to increase the amount of bioavailable Fe
in dehulled (decorticated) pulses (dal) such as lentil, in a biologically and culturally meaningful way,
to a level that could prevent Fe deficiency in humans. Our experimental approach had two main
objectives, first, to determine the most suitable iron fortificant and the appropriate dose of Fe for
dehulled lentil based on ease of fortification, and second, to determine the optimal processing
technology to fortify iron in dehulled lentil based on current processing practices. To fulfill the first
objective, research was focused on selection of the appropriate genotype and product type of dehulled
lentil, and identifying the best form of Fe solution with which to fortify dehulled lentil products. The Fe
fortificants, ferrous sulphate heptahydrate (FeSO
4·
7H
2
O), NaFeEDTA (ethylenediaminetetraacetic acid
iron (III) sodium salt) and ferrous sulphate monohydrate (FeSO
4·
H
2
O), are acceptable fortificants that

Nutrients 2017,9, 863 3 of 19
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.
2. 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.
2. Materials and Methods
The procedure followed for development of a lentil fortification protocol is shown in Figure 1,
and is discussed below.
Figure 1. 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).
2.1. 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 1. Flow chart for development of a lentil fortification protocol. aOven 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).
2.1. 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

Nutrients 2017,9, 863 4 of 19
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.
Nutrients 2017, 9, 863 4 of 19
(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 2. (a) Four dehulled, red lentil product types; (b) Fe concentration (ppm) in four dehulled,
unfortified, red lentil product types; and (c) Fe concentration (ppm) in red lentil product types
fortified with FeSO4·7H20 solution (400 ppm Fe). Different letters within each figure represent
significant differences (p < 0.05).
2.2. 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
FAO document “Guidelines on Food Fortification with Micronutrients” [13]. The FeSO4·7H2O and
FeSO4·H2O were supplied by Crown Technology, Inc., Indianapolis, IN, USA, and NaFeEDTA by
Figure 2.
(
a
) Four dehulled, red lentil product types; (
b
) Fe concentration (ppm) in four dehulled,
unfortified, red lentil product types; and (
c
) Fe concentration (ppm) in red lentil product types fortified
with FeSO
4·
7H
2
O solution (400 ppm Fe). Different letters within each figure represent significant
differences (p< 0.05).

Nutrients 2017,9, 863 5 of 19
2.2. 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].
2.3. Selection of an Appropriate Method of Fortification
2.3.1. 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 2 (Spray-Shake-Dry). Lentil dal was sprayed with fortificant solution using a 473 mL
clear, fine-mist spray bottle (SOFT
0
N STYLE, Product Code VO-302564, SKS Bottle and Packaging,
INC., Watervliet, NY, USA), shaken using a Barnstead Thermolyne M49235 Bigger Bill Orbital Shaker
(Sigma-Aldrich Corp., St. Louis, MO, USA) at 400 rpm for 10 min to mix the solution with the
dal sample, and subsequently dried to 14% moisture under a 250-watt electric heat lamp (NOMA
incandescent, clear, 130 V heat lamp, Trileaf Distributors, Toronto, ON, Canada) which produced
a temperature of approximately 70 ◦C at the surface of the fortified dal.
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.
2.3.2. 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.
2.3.3. 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

Nutrients 2017,9, 863 6 of 19
(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.
2.4. 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.
2.5. 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.
2.6. 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.

Nutrients 2017,9, 863 7 of 19
2.7. 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.
2.8. 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 M
Ω
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.
3. Results and Discussion
3.1. 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

Nutrients 2017,9, 863 8 of 19
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.
3.2. 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].
3.3. Selection of Appropriate Methods for Fortification
3.3.1. 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,

Nutrients 2017,9, 863 9 of 19
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.
3.3.2. 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.
3.3.3. 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].
Nutrients 2017, 9, 863 9 of 19
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 46.3–52.8, 25.3–33.1 and 36.6–
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.
3.3.3. 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 3. Iron concentration in polished football lentil dal fortified with FeSO4·7H2O solution (1600
ppm Fe) at 10 mL/100 g lentil dal using five different techniques. DSD = lentil dal oven dried for 10
minutes followed by soaking in fortificant solution and drying at 80 °C; SSD = lentil dal sprayed with
fortificant solution followed by shaking and drying; RDSD = lentil dal rinsed, oven dried, followed
by soaking in fortificant solution and then drying; SD = lentil dal directly soaked in fortificant solution
followed by drying; SRD = lentil dal soaked in fortificant solution followed by rinsing with deionized
water and drying. Different letters within the figure represent significant differences (p < 0.05).
Figure 3.
Iron concentration in polished football lentil dal fortified with FeSO
4·
7H
2
O solution
(1600 ppm Fe) at 10 mL/100 g lentil dal using five different techniques. DSD = lentil dal oven
dried for 10 minutes followed by soaking in fortificant solution and drying at 80
◦
C; SSD = lentil dal
sprayed with fortificant solution followed by shaking and drying; RDSD = lentil dal rinsed, oven
dried, followed by soaking in fortificant solution and then drying; SD = lentil dal directly soaked
in fortificant solution followed by drying; SRD = lentil dal soaked in fortificant solution followed
by rinsing with deionized water and drying. Different letters within the figure represent significant
differences (p< 0.05).

Nutrients 2017,9, 863 10 of 19
Nutrients 2017, 9, 863 10 of 19
Figure 4. (a) Fe-fortified lentil developed by five different fortification methods: SRD = lentil dal
soaked in fortificant solution followed by rinsing with deionized water and drying; SSD = lentil dal
sprayed with fortificant solution followed by shaking and drying; SD = lentil dal directly soaked in
fortificant solution followed by drying; RDSD = lentil dal rinsed, oven dried, followed by soaking in
fortificant solution and then drying; DSD = lentil dal oven dried for 10 minutes followed by soaking
in fortificant solution and drying at 80 °C; (b1–b4) Effect of different fortification methods on changes
in lightness (L*), yellowness (b*) and redness (a*) score of Fe-fortified lentil dal and on the fortification
process. Different letters within each figure represent significant differences (p < 0.05).
3.4. 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
Figure 4.
(
a
) Fe-fortified lentil developed by five different fortification methods: SRD = lentil dal soaked
in fortificant solution followed by rinsing with deionized water and drying; SSD = lentil dal sprayed
with fortificant solution followed by shaking and drying; SD = lentil dal directly soaked in fortificant
solution followed by drying; RDSD = lentil dal rinsed, oven dried, followed by soaking in fortificant
solution and then drying; DSD = lentil dal oven dried for 10 minutes followed by soaking in fortificant
solution and drying at 80
◦
C; (
b1
–
b4
) Effect of different fortification methods on changes in lightness
(L*), yellowness (b*) and redness (a*) score of Fe-fortified lentil dal and on the fortification process.
Different letters within each figure represent significant differences (p< 0.05).
3.4. 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

Nutrients 2017,9, 863 11 of 19
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.
Figure 5. pH of Fe solutions prepared with three fortificants (NaFeEDTA, FeSO4·7H2O, and
FeSO4·H2O) ranging in concentration from 200–3200 ppm. Different letters within each figure
represent significant differences (p < 0.05).
3.5. 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.
Fe Concentration in Fortificant Solution (ppm) Fe Concentration in Fortified Lentil Dal
FeSO4·7H2O NaFeEDTA FeSO4·H2O
Control 69.0 ± 0.9 a 69.0 ± 0.9 a 65.6 ± 0.8 a
100 76.0 ± 1.9 a 83.7 ± 2.5 a 71.8 ± 0.7 b
400 132.5 ± 3.2 b 113.2 ± 4.2 b 108.6 ± 1.1 c
800 147.9 ± 4.7 c 182.9 ± 5.8 c 151.4 ± 2.8 d
1200 157.8 ± 4.3 c 185.3 ± 5.6 c 185.0 ± 6.6 e
1600 203.6 ± 3.9 d 205.3 ± 2.8 d 207.5 ± 3.9 f
2000 217.5 ± 8.2 d 274.7 ± 5.6 e 261.8 ± 3.9 g
2400 246.6 ± 9.3 e 309.7 ± 10.0 f 322.3 ± 3.7 h
2800 286.7 ± 6.0 f 346.7 ± 5.2 g 363.5 ± 6.2 i
3200 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).
Figure 5.
pH of Fe solutions prepared with three fortificants (NaFeEDTA, FeSO
4·
7H
2
O, and FeSO
4·
H
2
O)
ranging in concentration from 200 to 3200 ppm. Different letters within each figure represent significant
differences (p< 0.05).
3.5. 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 to 3200 ppm.
Fe Concentration in Fortificant Solution (ppm) Fe Concentration in Fortified Lentil Dal
FeSO4·7H2O NaFeEDTA FeSO4·H2O
Control 69.0 ±0.9 a69.0 ±0.9 a65.6 ±0.8 a
100 76.0 ±1.9 a83.7 ±2.5 a71.8 ±0.7 b
400 132.5 ±3.2 b113.2 ±4.2 b108.6 ±1.1 c
800 147.9 ±4.7 c182.9 ±5.8 c151.4 ±2.8 d
1200 157.8 ±4.3 c185.3 ±5.6 c185.0 ±6.6 e
1600 203.6 ±3.9 d205.3 ±2.8 d207.5 ±3.9 f
2000 217.5 ±8.2 d274.7 ±5.6 e261.8 ±3.9 g
2400 246.6 ±9.3 e309.7 ±10.0 f322.3 ±3.7 h
2800 286.7 ±6.0 f346.7 ±5.2 g363.5 ±6.2 i
3200 349.0 ±1.8 g326 ±3.1 h381.7 ±3.6 j
a
Mean
±
SD. Mean scores for Fe concentration followed by different letters within columns are significantly
different (p< 0.001).

Nutrients 2017,9, 863 12 of 19
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.
Fe Concentration (ppm)
Lightness (L*) Redness (a*) Yellowness (b*)
Initial After 6 Months After One
Year Initial After 6 Months After One
Year Initial After 6 Months After One
Year
FeSO4·7H2O fortified samples
Control 50.6 ±0.4 a50.8 ±0.2 a51.0 ±0.2 a31.5 ±0.2 a31.3 ±0.2 a30.6 ±0.6 a41.6 ±1.0 a41.2 ±1.0 a40.3 ±1.0 a
200 49.9 ±0.6 ab 50.6 ±0.6 a52.0 ±0.5 b29.7 ±0.8 b29.4 ±0.8 b28.8 ±0.8 b40.5 ±0.1 b38.9 ±0.1 b37.9 ±0.1 b
800 49.6 ±0.2 b50.3 ±0.1 a51.5 ±0.0 b27.4 ±0.3 c26.8 ±0.2 c
25.8
±
0.3
ac 37.8 ±0.3 c36.4 ±0.3 c34.6 ±0.3 c
1600 46.2 ±0.5 c46.9 ±0.5 b48.5 ±0.4 c24.6 ±0.7 d 24.9 ±0.6 d25.5 ±1.2 c36.4 ±0.1 d33.9 ±0.1 d34.0 ±0.1 c
2400 43.9 ±0.2 d44.5 ±0.1 c45.8 ±0.2 c22.6 ±0.2 e22.2 ±0.1 e21.3 ±0.2 d32.0 ±0.3 e31.2 ±0.1 e30.0 ±0.4 d
3200 42.1 ±0.6 e42.7 ±0.6 d43.9 ±0.6 d21.3 ±0.8 f34.4 ±0.9 f20.3 ±1.2 d30.0 ±0.2 f29.7 ±0.7 f28.6 ±0.3 e
NaFeEDTA fortified samples
Control 50.5 ±0.4 a50.8 ±0.2 a50.8 ±0.2 a31.5 ±0.2 a31.3 ±0.3 a30.6 ±0.6 a41.6 ±0.3 a41.2 ±0.1 a40.3 ±0.7 a
200 50.4 ±0.1 a51.0 ±0.2 a51.0 ±0.2 a31.6 ±0.7 a31.1 ±0.8 a30.3 ±0.8 a41.9 ±0.1 a41.5 ±0.1 a40.6 ±0.3 a
800 50.1 ±0.2 a50.6 ±0.6 a50.6 ±0.6 b31.1 ±0.3 a30.5 ±0.2 a29.0 ±0.5 b40.6 ±0.9 b39.3 ±0.4 a36.9 ±0.8 b
1600 48.8 ±0.1 b52.0 ±0.5 b52.0 ±0.5 b29.4 ±0.3 b29.1 ±0.2 b28.6 ±0.4 b38.9 ±0.2 c38.2 ±0.2 b36.6 ±0.5 b
2400 47.5 ±0.2 c50.3 ±0.1 c50.3 ±0.1 c27.5 ±1.3 c27.0 ±1.2 c26.1 ±1.1 c36.3 ±0.7 d35.8 ±0.6 c34.6 ±0.6 c
3200 46.4 ±0.5 d51.5 ±0.0 d51.5 ±0.0 c27.8 ±0.4 c27.4 ±0.4 c26.5 ±0.4 c36.9 ±0.7 d36.4 ±0.8 c35.2 ±0.9 c
FeSO4·H2O fortified samples
Control 50.5 ±0.4 a50.5 ±0.4 a50.8 ±0.2 a51.2 ±0.3 a31.5 ±0.2 a31.3 ±0.2 a30.6 ±0.6 a41.6 ±0.3 a41.2 ±0.1 a
200 51.1 ±0.5 a51.1 ±0.5 a51.3 ±0.3 b51.7 ±0.3 b30.0 ±0.7 b29.9 ±0.7 a29.8 ±0.7 b39.9 ±0.1 b39.6 ±0.1 b
800 49.3±0.7 b49.7 ±0.7 b50.4 ±0.5 b27.9 ±0.3 c27.6 ±0.4 c27.1 ±0.4 a37.3 ±0.9 c36.9 ±0.4 c36.5 ±0.8 c
1600 46.9 ±0.7 c47.3 ±0.4 c48.1 ±0.2 c25.4 ±0.3 d25.4 ±0.3 d25.4 ±0.4 c34.6 ±0.2 d34.6 ±0.2 d34.6 ±0.5 d
2400 44.4 ±0.6 d44.7 ±0.4 d45.4 ±0.4 d23.3 ±0.7 e22.8 ±0.7 e21.9 ±0.9 d32.2 ±0.7 e31.9 ±0.6 e30.2 ±0.6 e
3200 42.6 ±0.3 e42.6 ±0.3 e42.7 ±0.5 e22.7 ±0.7 e22.1 ±0.7 e21.1 ±0.8 d31.5 ±0.7 e30.9 ±0.8 f29.8 ±0.9 f
aMean ±SD. Mean scores for lightness (L*), redness (a*) and yellowness (b*) score followed by different letters within columns are significantly different (p< 0.001).

Nutrients 2017,9, 863 13 of 19
3.6. 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].
3.7. 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 FeSO4·7H2O or FeSO4·H2O (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.

Nutrients 2017,9, 863 14 of 19
Nutrients 2017, 9, 863 14 of 19
Figure 6. Effect of increasing Fe concentration on lightness (L*), redness (a*) and yellowness scores
(b*) of lentil dal samples fortified with FeSO4·7H2O, NaFeEDTA and FeSO4·H2O at five different
concentrations ranging from 200–3200 ppm. Different letters within each figure represent significant
differences (p < 0.05).
Figure 6.
Effect of increasing Fe concentration on lightness (L*), redness (a*) and yellowness scores
(b*) of lentil dal samples fortified with FeSO
4·
7H
2
O, NaFeEDTA and FeSO
4·
H
2
O at five different
concentrations ranging from 200 to 3200 ppm. Different letters within each figure represent significant
differences (p< 0.05).

Nutrients 2017,9, 863 15 of 19
Nutrients 2017, 9, 863 15 of 19
Figure 7. Effect of storage time on changes in L*, a* and b* score of football lentil samples fortified
with 1600 ppm of Fe using FeSO4·7H2O, NaFeEDTA and FeSO4·H2O. Different letters within each
figure represent significant differences (p < 0.05).
3.8. 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.
3.9. 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
Figure 7.
Effect of storage time on changes in L*, a* and b* score of football lentil samples fortified with
1600 ppm of Fe using FeSO
4·
7H
2
O, NaFeEDTA and FeSO
4·
H
2
O. Different letters within each figure
represent significant differences (p< 0.05).
3.8. 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-,
FeSO4·H2O- or NaFeEDTA-fortified samples compared to the control.

Nutrients 2017,9, 863 16 of 19
3.9. 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.
Cooked Lentil Sample
Fe
Concentration
(ppm) a
Ferritin Formation
(ng Ferritin (mg
Protein)−1)a
Relative Fe
Bioavailability (%
Control Lentil) a
Phytic Acid
(mg g−1)a
Unfortified dehulled lentil 68.7 ±0.3 a12.7 ±1.0 a68.3 ±14.8 a8.0 ±0.1 a
NaFeEDTA fortified (1600 ppm Fe) 230.8 ±8.5 b17.4 ±2.7 b100.5 ±7.5 b8.0 ±0.2 a
FeSO4·H2O fortified (1600 ppm Fe) 220.5 ±2.1 c17.6 ±2.2 b104.9 ±16.7 b7.2 ±0.1 c
FeSO
4·
7H
2
O fortified (1600 ppm Fe)
238.5 ±4.7 b21.2 ±1.9 b103.4 ±10.4 b7.4 ±0.1 b
a
Mean
±
SD. Mean scores for Fe concentration, bioavailability (ng ferritin (mg protein)
−1
), relative Fe bioavailability
(% control lentil) and phytic acid (mg g
−1
) followed by different letters within columns are significantly different
(p< 0.001).
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.
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).
Acknowledgments:
The authors would like to acknowledge financial assistance received from The Saskatchewan
Ministry of Agriculture (Agriculture Development Fund) and Grand Challenges Canada. The authors are grateful
for technical assistance provided by B. Goetz, Crop Development Centre, University of Saskatchewan and
Chowdhury Jalal, Micronutrient Initiative, Ottawa for invaluable guidance during manuscript preparation.
Author Contributions:
R. Podder and A. Vandenberg conceived and designed the study. R. Podder, B. Tar’an
and D. DellaValle analysed the data. R. Podder prepared the draft manuscript. B. Tar’an, R. T. Tyler, C. J. Henry,
D. M. DellaValle and A. Vandenberg reviewed all documents critically and approved the final manuscript for
submission in the Journal.
Conflicts of Interest: The authors declare no conflict of interest.

Nutrients 2017,9, 863 17 of 19
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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