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
]. Lentil is a good source of non-heme iron, ranging from 73 to 90 mg kg−1
]. 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
]. 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
Iron (Fe) is the most abundant element in the earth′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 (FeSO4
O), NaFeEDTA (ethylenediaminetetraacetic acid iron (III) sodium salt) and ferrous sulphate monohydrate (FeSO4
O), are acceptable fortificants that 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.
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 2
a). 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
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
O, NaFeEDTA and FeSO4
O were selected from a list of iron fortificants published in the WHO and FAO document “Guidelines on Food Fortification with Micronutrients” [13
]. The FeSO4
O and FeSO4
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 FeSO4·7H2O, one of the three Fe fortificants studied. Five methods were used to fortify lentil dal with FeSO4·7H2O 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 ′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 FeSO4·7H2O 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.
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 HNO3
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 HNO3
at 90 °C for 45 min, followed by addition of 5 mL of 30% H2
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
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 H2O 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.
2.7. Boiling Time Estimation of Fortified Lentil Dal Samples
Three fortified dal samples (FeSO4
0, NaFeEDTA and FeSO4
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 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 HNO3
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
], 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