Iron and Folate Contents of Tajik Legumes

Seven varieties of Tajik legumes and two Tajik snack type ready-to-eat (RTE) whole/split chickpeas were analyzed for iron in raw and cooked legumes and for naturally occurring folate content in cooked legumes. Iron was measured according to AACC method 40 41B. Folate contents were determined by microbiological (Lactobacillus casei subsp. Rhamnosus ATCC 7469) and high-performance liquid chromatography analysis utilizing a tri-enzyme treatment (protease, α-amylase and conjugase). Folate derivatives of tetrahydrofolate, 5-formyl-tetrahydrofolate and 5-methyl-tetrahydrofolate were identified and quantified. Iron content for Tajik legumes ranged from 5.52 to 13.27 mg/100 g for raw; 2.81 to 4.12 mg/100 g for cooked and 4.37 and 4.76 mg/100 g for RTE chickpeas. The total folate content of cooked legumes ranged from 53 to 81 μg/100 g for beans; 133 to 203 μg/100 g for peas, and from 39 to 22 μg/100 g for small and large lentils, respectively. The predominant form of folate in legumes was tetrahydrofolate, followed by 5-formyl-tetrahydrofolate and 5-methyl-tetrahydrofolate.


Introduction
Vitamin and mineral deficiencies (also known as "hidden hunger") are particularly prevalent in Central Asia with a high prevalence of iron and folate deficiency [1].Anemia is the most common blood disorder in adults [2].Both iron and folate deficiencies contribute to the prevalence of anemia [3].The annual estimate of iron-deficiency anemia is 42% among Tajik women between 15 -49 years old; approximately 100 maternal deaths per year occur from severe anemia [4].In the geographic region surrounding Dushanbe (the capital of Tajikistan), but not including the capital, a high rate (73.7%) of folic acid deficiency in women was found [5].In Tajikistan, over 300 children annually are born with neutral tube defects [4].Iron intake from animal products was negligible among Tajik women [5].Folate intake from consumption of green leafy vegetables was 15.6%; 11.6% from melons and pumpkins; and 1.5% from fruits and berries [5].
A food-based approach to reduce micronutrient malnutrition in Tajikistan encourages dietary diversification through the production and consumption of micronutrient-rich foods, including appropriate traditional foods [6].This strategy is cost-effective, sustainable and income generating; culturally acceptable and feasible to implement; and builds alliances among government, consumer groups, the food industry and other relevant organizations to achieve the shared goal of preventing micronutrient malnutrition [7].
Legumes are grown for direct human consumption, available all year, and important sources of dietary proteins, dietary fiber, iron, folate, zinc, and calcium [8,9].Legumes can be milled into flour to make bread and bread products and extruded snacks [10].In Tajikistan, chickpeas are minimally processed into a snack food which is consumed like peanuts in the US.Legumes are an inexpensive source of nutrients and are culturally acceptable both in the diet and as a crop in Tajikistan [11].The joint FAO/WHO expert consultation on nutrition and mineral requirements states that pulses (legumes), vegetables, including green leafy vegetables and fruits are the preferred way of ensuring optimal nutrition, including micronutrient adequacy [12].
Nutritional assessment by diet analysis of a population includes the evaluation of food consumed and then converting these food items into their nutrients [13].This process requires a food composition database which lists the nutritional values for a given food portion for foods commonly eaten by the population [13].
Therefore, the objectives of our study were to 1) evaluate the iron content of raw and cooked Tajik legumes by the orthophenanthroline spectrophotometry; and 2) determine the naturally occurring folate contents of cooked Tajik legumes and ready-to-eat snack type chickpeas by microbiological and high-performance liquid chromatography (HPLC) methods.

Cooking Treatments
Fifty g of legumes were soaked in 150 ml distilled water at 4˚C for 16 h.The soaked legumes were held at 4˚C to minimize microbial growth.After 16 h, the soaking water was discarded, 250 ml distilled water was added to the soaked legumes and legumes were then cooked until doneness on an electric hot plate.Doneness was determined visually and subjectively by pressing between the fingers (approximate cooking times for beans, peas and lentils were 30, 20 and 10 min).Cooked legumes were drained before analysis and stored at -80˚C for iron and folate determination.Weights of legumes before and after cooking were recorded.Each variety of raw and cooked legumes was analyzed in triplicate for iron measurements; for folate measurements, cooked legumes were analyzed in triplicate.

Iron Measurements
The AACC method 40 -41B [14] was used for the determination of the iron content.Iron reference solution and 1, 10-phenanthroline monohydrate (Cl 12 H 8 N 2 •H 2 O) were obtained from Fisher Scientific (Fair Lawn, NJ, USA) and hydroxylamine hydrochloride (NH 2 OH • HCl) from ACROS Organics (Geel, Belgium).Legumes were homogenized in an Osterizer™ blender.A 5 g sample of raw or cooked legumes were dry ashed and dissolved in 5 ml of concentrated HCl and diluted to 100 ml.A 10 ml of the aliquot was taken for reaction with 1 ml of NH 2 OH•HCl solution and 1 ml of orthophenanthroline.The absorbance of each solution was measured spectrophotometrically (Beckman DU-640, Fullerton, CA) at 510 nm and converted to iron concentration (mg/ml) using a calibration curve.
True Retention True retention of iron content was calculated as described by Murphy et al. [15] using the following formula: True retention (%) = [(nutrient content (g/100 g) of cooked legume × g of cooked legume)/(nutrient content (g/100 g) of raw legume × g of raw legume) × 100].

Microbiological Analysis
Microbiological analysis of the total folate contents was based on Rader et al. [16] with a modification to the amount of the conjugase source.All procedures were carried out under subdued light conditions.A 25 g sample of cooked legumes was homogenized in 100 ml of 0.1 M phosphate buffer (pH 7.0) and centrifuged for 30 min.A 1 ml (2 mg•ml -1 ) of protease (Streptomyces griseus; Sigma Chemical Co., St. Louis, MO) was added to 25 ml extract and incubated for 3 h at 37˚C.Samples were heated to 100˚C, cooled and brought to pH 7.8 with buffer.A 1 ml (20 mg•ml -1 ) of α-amylase (Aspergillus oryzae; Sigma Chemical Co., St. Louis, MO) was added to each extraction sample and incubated for 2 h at 37˚C.A 4 ml (3 mg•ml -1 ) aliquot of chicken conjugase (Difco Laboratories, Detroit, MI) was added to each extraction sample and incubated for 16 h at 37˚C.Deconjugated

Sample Extraction and Purification
After protease treatment (as described above), the pH was adjusted to 6.8.Rat plasma (Difco Laboratories, Detroit, MI) at a concentration of 0.5 ml was used in 10 ml of extract as a conjugase source.The extracts were purified by solid phase extraction (SPE) on a strong anion exchange (SAX) cartridges [Quaterny amine (N + ), Baker 7091-3] as described by Osseyi [18].The cartridges (3-ml SPE column, 500 mg) were conditioned with 3 ml of hexane, followed by 3 ml of methanol, and then equilibrated with 5 ml of 0.

Quality Control
Cooked legume samples were spiked with a known amount of folic acid and analyzed by microbiological and HPLC methodologies.Recovery was calculated using the following formula: % Recovery = [(ng folic acid in spiked sample -ng folic acid in unspiked sample)/ng folic acid added in spiked sample.Recovery of added folic acid averaged 93% -102%.

Statistical Analysis
For all analysis, the means of three values and standard errors were calculated using statistical analysis software (SAS Institute, Inc., Cary, NC, 2000; Release 8.01).A level of 5% was considered to be significant.

Iron Content
Seven varieties of Tajik legumes and two Tajik snack type ready-to-eat (RTE) whole/split chickpeas were analyzed for iron content (Table 1).The effect of cooking on the true retention for iron in legumes is presented in Table 2.For true retentions of less than 100%, the iron may have been lost in the soaking or cooking water.Murphy et al. [15] also reported iron retention greater than 100% in legumes and attributed this increase in foods where moisture is gained during soaking/cooking and the potential of solids are lost during cooking.In our study, the cooking water was not analyzed for iron content.
Results of analyzed iron contents of Tajik legumes were compared with the USDA Nutrient Database [19].Tajik legumes were 35% lower for raw and 56% higher for cooked legumes than the USDA Nutrient Database [19] (Table 2).These differences may be due to varietal differences, growing conditions, storage conditions and preparation methods.Also, the soaking and cooking process that we used may have affected the higher retentions of iron in the Tajik varieties.
In our study, legumes were soaked overnight for 16 h.Soaking legumes for some period of time is one of the common practices of Tajik households.Soaking improves iron absorption, which degrades the phytate [20].However, according to Brazaca and Da Silva [21], the effects of tannins and phytic acid were not determining factors in diminishing the amount of dialyzed iron; rather the effect of dietary enhancers (meat and orange juice) increased the amount of iron absorption.Therefore, in addition to soaking, including foods rich in vitamin C (tomatoes, leafy green vegetables, bell peppers, lemon, and green onions) and/or small amount of meat to a cooked legume item may help maximize iron absorption.

Folate Contents
The same varieties of cooked legumes were analyzed for the folate contents by microbiological analysis and HPLC.In general, the microbiological analysis is based on quantifying the growth response of a specific microorganism, L. casei subsp.Rhamnosus, to the total mixture of folate that are present [22,23].The use of the HPLC analysis allows the identification of the boilogically active forms of folate; tetrahydrofolate (THF), 5--methyl tetrahydrofolate (5-CH 3 -THF), and 5-formyl tetrahydrofolate (5-CHO-THF).
Legumes are rich in protein and starch; therefore, the complete extraction of folates from the cellular matrix of legume seed is essential.In our study, the tri-enzyme treatment was used, including protease, α-amylase and conjugase [16].Chicken pancrease was used as a conjugase source for microbiological analysis and rat plasma for HPLC.The total folate contents by microbiological analysis of cooked legumes ranged from 53 to 81 µg/100 g for beans; 133 to 203 µg/100 g for peas, and from 22 to 39 µg/100 g for lentils (Table 3).RTE whole or split chickpeas prepared and used as a snack food in Tajikistan had a high folate content (269 and 233 µg/100 g) on a dry basis, respectively (Table 3).Our results were simi-  lar to folate contents of cooked legumes, soaked overnight for 16 h, reported by Hoppner and Lampi [24].However, the total folate contents were lower than those reported in the USDA Nutrient Database (Table 3).Measurement of folate contents by different extraction methodologies, as well as the nature of the growing conditions, weather, location and cultivar has an impact on food components [25][26][27][28] storage conditions, legumes may become dry and cracked, which may influence the loss of vitamins from outer layers during soaking, as most B vitamins are located in the outer layers.
Seven varieties of cooked legumes analyzed by HPLC contained 49.9% of folates in the form of THF; 29.5% in the form of 5-CHO-THF; and 20.6% in the form of 5-CH 3 -THF (Table 3).RTE chickpeas contained mainly 95% of folates in the THF form (Table 3).The distributions of folates derivatives in kabuli type of chickpeas (Table 3) were higher (28.1 µg/100 g for 5-CH 3 -THF, 15.3 µg/100 g for 5-CHO-THF, 51.4 µg/100 g for THF) than the results of Ruggeri et al. [27]; 9.1 µg/100 g for 5-CH 3 -THF, 6.1 µg/100 g for 5-CHO-THF and not detectable for THF.Another researcher, Rychlik et al. [29] determined the folate content of legumes using a stable isotope dilution assays and found that 5-CH 3 -THF was the predominate form in frozen peas, dried lentils, dried blackeye peas and mung beans; THF was the most abundant in fresh beans and soybeans; and 5-CHO-THF was predominately found in peanuts.
The mean differences of folate as measured by the HPLC analysis were approximately 29% lower than the total folate amounts analyzed by microbiological analysis.Differences may be due to the identification of only three folate forms (THF, 5-CH 3 -THF, and 5-CHO-THF) by HPLC analysis that are found in food; therefore, not including other folate compounds which may be found in minute quantities and not measured.The higher total folate values that we found as measured by the microbiological analysis may be due to non-folate compounds that stimulate bacterial growth [30].
In our study, we did not analyze folate in raw legumes.Folate losses may occur through leaching (soaking for 16 h) and cooking.Leaching of folates also depends on the type and variety of legumes [35], size and the surface area to volume ratio during soaking [24].In our study, for example, small lentils were higher in total folate values (39 µg/100 g) compared to large lentils (22 µg/100 g); and desi type chickpeas (small) had higher total folate content (203 µg/100 g) compared to kabuli type chickpeas (large) which contained 189 µg folate/ 100 g.
Differences of folate forms from our study compared to other research may be attributed to discarding the soaking and cooking water rather than the destruction of folate forms during cooking [24,36].However, legumes soaked overnight for 16 h have been shown to have higher folate retentions compared to no presoak or quick soak treatments [24].Folate content of the cooking water was not determined in our study.It has been suggested to use the cooking water from legumes for further consumption [36].In Tajikistan, the most common way of preparing legumes is cooking the legume and using the water as broth for stew, soup or other food items.

Conclusions
Iron and folate contents of Tajik varieties of cooked legumes could be used in dietary research and to make recommendations to Tajiks (especially women of childbearing age) to improve their iron and folate status.Using our data and calculations, the dietary iron intake for Tajik women (18 or above) consuming 100 mg of cooked legumes would have an iron intake of 18% from cranberry and red kidney beans, 17% peas, 28% lentils, and 25% from RTE chickpeas, based on 18 mg of iron per day as advised by Dietary Reference Intakes.The dietary folate intake for Tajik women (18 or above) consuming 100 g of cooked legumes would have a folate intake of 17% from cranberry and red kidney beans, 44% peas, 8% lentils, and 63% from RTE chickpeas, based on 400 µg of folate.

Table 1 . Description of legumes grown and purchased in Tajikistan.
Legumes DescriptionRed, kidney beans (Phaseolus vulgaris) -large, kidney-shaped bean; deep reddish-brown; robust, full-bodied flavor Cranberry beans (Romano) -large ovals; creamy background with burgundy highlights, pink-colored Blackeye peas (Vigna unguiculata) -kidney shaped white bean with black eye Kabuli type chickpeas (Cicer arietinum) -round, beige color; nut-like flavor ad firm texture Desi type chickpeas (Cicer arietinum) -angular, pigmented seeds, beige color; nut like flavor and firm texture Lentil, large (Lens culinaris) -seeds are round, about ½ cm in diameter and light green in color Lentil, small (Lens culinaris) -seeds are round, brown in color mixed with dark brown seeds speckled with black Chickpeas whole (RTE) (Cicer arietinum) -round, medium size, about ½ cm in diameter and light green in color Chickpeas split (RTE) (Cicer arietinum) -round, split, yellow color; nut-like flavor and firm texture 3 -THF, folic acid were purchased from Sigma Chemical Co.(St.Louis, MO).