Evaluation of anti-nutrient and mineral content in different pearl millet varieties

Pearl millet is one of the most popular crops in India. It is nutritionally comparable and even superior to major cereals with respect to protein, fat, energy, vitamins & minerals. The present investigation was carried out to evaluate the mineral content and the anti-nutrients present in pearl millet varieties i.e., white grain variety (ICMV-221), hybrids varieties (HHB-226, HHB-223 and HHB-197) and HC-20 variety. Calcium content was highest in HHB-226 and lowest in ICMV-221. No significant differences in calcium content of HHB-197 and HHB-223 was witnessed. Significant (P< 0.05) differences in iron content of different varieties of pearl millet were observed. ICMV-221 contained significantly (P< 0.05) higher zinc content than all the other varieties of pearl millet however, no significant difference in zinc content of HC-20 and HHB-223 as well as HHB-197 and HHB-226 were noticed. The phytic acid and polyphenol content of all pearl millet varieties varied from 620.00 to 875.00 and 304.17 to 438.33 mg/100g.


Introduction
Changing life style, changes in meal pattern and awareness about health as well as healthy foods has increased the demand for consumption of the high content of minerals present in cereals. In this category, more emphasize is on Pearl millet [Pennisetum glaucum (L.)] which is the fourth most important grain crop next to rice, wheat and corn. Moreover, pearl millet is nutritionally comparable and even superior to major cereals with respect to protein, fat, energy, vitamins & minerals (Hadimani et al., 1995;Abadalla et al., 1998) [9,3] . It also has better mineral profile because of higher level of calcium but owing to certain inherent factors, the availability of these mineral to human system is low (Kumar and Kapoor, 1984) [11] . 6. 0.1 N Potassium permanganate (KMnO4) 7. 0.1N Potassium permanganate: Dilute 10 ml of 0. 1N Potassium permanganate solution to 100 ml with water (1 ml = 0.2 mg of Ca). Prepare fresh solution before use.

Procedure
Twenty ml of the digested mineral solution was taken in a 250 ml beaker. 25 ml of water was added to it. 10 ml of saturated ammonium oxalate solution and 2 drops of methyl red indicator was added to the ash solution. Solution was made alkaline by adding dilute ammonia and then slightly acidic with a few drops of acetic acid until the colour was faint pink (5.0 pH). The solution was heated to boiling and was allowed to stand at room temperature for overnight. The solution was then filtered through Whatmann No. 42 paper and washed with water, till the filterate was oxalate free. The point of filter paper was broken with platinum wire or glass rod. Then the precipitates were washed with hot dilute sulphuric acid from wash bottle into beaker in which the calcium was precipitated and then with hot water and titrated hot (temperature 70-80 °C) with 0.01N Potassium permanganate (KMnO 4 ) till permanent pink color.
Where X = Titre value V = Total volume of ash solution V 1 = Volume taken for estimation W = Weight of sample

Total dietary fibre
The total dietary fibre was determined by slight modification of an enzymatic method using AOAC method (1995) [5] . Reagents: 1. 78% ethanol (207 ml of water was taken into one liter volumetric flask and 95% ethanol was added to make one liter. 2. Phosphate buffer: 0.08 M, pH 6.0-6.5. 1.4 g of Na 2 HPO 4 and 8.4 g of NaH 2 PO 4 anhydrous was dissolved in one liter of water. The pH was checked and adjusted with NaOH. The buffer made was stored in tightly capped container at room temperature. 3. 0.275 N NaOH. Diluted 275 ml of 0.1N NaOH solution to one liter with water in a volumetric flask and stored in tightly capped container at room temperature. 4. 0.325 M HCl. Diluted 325 ml of 1.0N HCl solution to one liter with water in a volumetric flask and stored in tightly capped container at room temperature. 5. Enzymes -α-amylase, protease and amyloglucoside obtained from Sigma Aldrich Inc. Pvt. Ltd.

Procedure
Sample preparation: one g sample (S) was weighed and defatted it in Soxhlet apparatus.
Extraction of water soluble material: The sample was dispersed in a 50 ml of pH 6.0 phosphate buffer and was kept for 40 min. at 60 0 C to ensure interaction of pectin with minimum degradation.

Starch and protein hydrolysis:
The pH of solution was adjusted to 6.0-6.5 for optimum amylase and protease activity. The suspension was then cooled to 20-30 º C. Then add 0.10 ml α-amylase was added to the beaker and mixed well. The beaker was covered with aluminum foil and placed in a boiling water bath. Beaker was agitated gently at 5 minutes interval, temperature of the beaker reached at 95 º C. Solutions were allowed to cool at room temperature. The pH of the solution was adjusted to 7.5+0.2 by adding 10 ml of 0.275 N NaOH to beaker. The pH was checked and adjusted with NaOH. Solution of protease in phosphate buffer (5 mg/ml) was prepared fresh. 0.1 ml of 5 mg protease solution was added to beaker. The pH of the solution was adjusted between 4.0 and 4.6 by the addition of 10 ml of 0.325 M HCL to beaker, pH was checked and adjusted with NaOH, 0.1 ml of amyloglucoside was added to beaker and covered it with aluminum foil. Then place in 60 0 C water bath, with continuous agitation; incubated for 30 minutes after the internal temperature of the beaker reached 60 0 C. Four volumes of 95% ethanol added to beaker. The solution was kept overnight at room temperature for complete precipitation.
Filtration: After complete precipitation, the bed of celite in crucible was wetted and redistributed by using 78% ethanol. Gentle suction was maintained and quantitatively transferred the precipitate and suspension from beaker to the crucible.
The residue was washed with three 20 ml portions of 78% ethanol, two 10 ml portions of 95% ethanol and two 10 ml portions of acetone. The crucible containing residues was dried overnight in a 105 o C air oven. The crucible was cooled in desiccators and the weight as "residue + celite + crucible weight" (R) was recorded. The crucible was transferred to muffle furnace and heated to 525 °C 5 hours. After complete ashing, crucible was cooled in desiccators. The weight was recorded i.e. weight of "ash + celite + crucible weight" (A).

Procedure
Extraction of sample: one g sample was extracted separately with distilled water and 80% ethanol by refluxing for 1hour and then centrifugation was carried out at 2000 rpm for 30 minutes. After filtration, the residue was re-extracted twice under same conditions. The solvent was removed to get extractives. These extract were filtered and concentrated by keeping it in oven. The total phenolics were determined by Folin-Ciocalteu reagent method using Gallic acid as standard for which a calibration curve was obtained with solutions 0.08, 0.04, 0.02, 0.01 and 0.05 mg/ml of Gallic acid. A 1.0 ml of diluted extract (all fraction were diluted with ethanol to adjust the absorbance with the calibration limits), 1.0ml of 1mol/L Folin-ciocalteau reagent and 2.0 ml of Na 2 CO 3 (20% w/v) were mixed and the volume was made to 10 ml. After 8 minutes, the mixture was centrifuged at 600 rpm for 10 minutes. Then the absorbance of supernatant solution was measured at 730 nm using Spectronic 20 (Elico Ltd. Hyderabad) spectrophotometer and against a blank prepared similarly but containing distilled water instead of extract. The concentration of phenolics thus obtained was multiplied by the dilution factor and the results were expressed as the equivalent to milligrams of Gallic acid per gram of extract (mg GAE/g).

Conclusion
The study showed that calcium content was highest in HHB-226 and lowest in ICMV-221. HC-20 contained highest iron content followed by ICMV-221, HHB-197, HHB-223 and HHB-226. Highest zinc content was found in white grain variety (ICMV-221) whereas lowest in HHB-226 variety of pearl millet. Lowest phytic acid content was found in white grain variety whereas HHB-223 contained highest phytic acid. However, lowest polyphenol content was in HHB-223 variety of pearl millet whereas highest polyphenol content was noticed in HC-20. Thus, all above mentioned pearl millet varieties can be utilized for processing and to prepare iron, calcium rich healthy foods and also help in diversifying its use for achieving food and nutritional security.