Effect of malting period on physicochemical properties, minerals, and phytic acid of finger millet (Eleusine coracana) flour varieties

Abstract Background Deficiency of essential minerals is a widespread nutritional disorder in the world, particularly in developing economies. Poor mineral accessibility from foods is a major contributing factor to deficiency and associated health problems. This study investigated the effect of malting on minerals, phytic acid, and physicochemical properties of finger millet varieties. Sorghum was used as external reference. Mineral composition was analyzed using an inductively coupled plasma atomic emission spectroscopy (ICP‐AES) and mass spectroscopy (ICP‐MS). Results Data showed that finger millet is rich in macroelements and trace elements. Malting for 24 hr reduced mineral content of the grains except sodium. Increase in the minerals was observed beyond 48 hr of malting particularly at 96 hr. Successive decrease in phytic acid of the grains was not observed with malting time. Malting did not result in any significant change in the physicochemical properties of the grains. Conclusion ICP‐AES/MS showed that finger millet contain a variety of minerals in amounts that were not previously reported, and malting the grain for 72 to 96 hr positively affected the minerals. Changes in phytic acid suggest that phytate undergoes dissociation during malting rather than a degradation of phytic acid. Potential exists for utilization of finger millet as functional ingredient to augment important minerals in weaning, geriatric, and adult foods for health promotion.


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UDEH Et al. due to minimal inclusion in ready-to-use or ready-to-eat convenience food products, unawareness by the general population, lack of research, and novel product development processes (Kumar et al., 2016;Radchuk et al., 2012;Shahidi & Chandrasekara, 2013). The very small size and subsistent scale of production are also a significant technological setback to utilization.
A number of certain inorganic minerals are essential for numerous biological activities which are important for human performance and survival and therefore should be provided in the diet. They constitute a large part of the body intracellular fluid where they act as cofactors to several enzyme systems, neuronal transmission, muscle contraction, and ionic balance. Beside their bodily functions, studies have shown that they play an important role in the treatment of metabolic conditions such as diabetes, hypertension, cardiovascular disorder, anemia, osteoporosis, and immune abnormalities among others (Fraga, 2005;Goldhaber, 2003;Sasaki et al., 1999;World Health Organisation and Food and Agriculture Organisation of the United Nations (WHO/FAO), 2004). Deficiency of essential minerals such as calcium, potassium, magnesium, iron, and zinc are common, and widespread nutritional disorder in the world particularly in developing economies (Platel, Eipeson, & Srinivasan, 2010;WHO, 2001;WHO/FAO, 2004). Another major concern of deficiency of important minerals is in gluten-free diets. Gluten-free foods which are an important trend in the nutrition of people who are allergic to gluten or wheat, and in other healthy individuals who follow gluten free foods, have also been shown to contain less valuable minerals than gluten containing food products (Goldhaber, 2003). Poor accessibility of minerals from plant food is a major contributing factor to their deficiency and associated health problems (Fraga, 2005). In cereal grain, particularly, mineral availability is associated with antinutritional factors like phytic acid and polyphenols that form complexes with the minerals thus hindering their release.
Finger millet is considerably rich in minerals, and its micronutrient density is higher than that of major cereal grains: rice and wheat (Kumar et al., 2016). Its calcium and iodine content is reported to be the highest among cereals (Reddy et al., 2008). The nutrient-rich grain is mainly used for making unleavened bread among other variety of preparations such as cakes, puddings, porridge, etc. The grain malt flour are used in the preparation of infant and geriatric foods, and as a popular food supplement for diabetics (Kumar et al., 2016;Reddy et al., 2008). Finger millet is gluten-free and hence is good for patients suffering from celiac disease (Pagano, 2006;Saturni, Ferretti, & Bacchetti, 2010). It has been recently used as composite flour for making biscuit due to its high calcium content (Krishnan, Dharmaraj, & Malleshi, 2012).
However, due to its high phytate (Afify, El-Beltagi, El-Salam, & Omran, 2011;Jha, Krishnan, & Meera, 2015;Mamiro, Van Camp, Mwikya, & Huyghebaert, 2001;Sripriya, Anthony, & Chandra, 1997) and polyphenol (Krishnan et al., 2012;Tako, Reed, Budiman, Hart, & Glahn, 2015) content, the availability of minerals in finger millet is low thus presenting ground for concern. A study by Platel et al. (2010) reported that malting had no significant influence on the mineral content of finger millet except for wheat where a slight decrease in copper content was observed. Mamiro et al. (2001) did not find any significant change in calcium, zinc, and iron content of soaked and germinated finger millet. The report of Sripriya et al. (1997) showed that germination was effective in increasing trace elements such as copper, zinc, and manganese while fermentation was more effective in increasing calcium, phosphorus, and iron from finger millet. Information on the mineral composition of finger millet and how they are affected by malting period and phytic acid content is relatively scarce and inconclusive, and thus requires further elucidation. Here we report the application of inductively coupled plasma atomic emission spectroscopy (ICP-AES) and mass spectroscopy (ICP-MS) as a tool to study the minerals present in finger millet grain. Changes in the physicochemical properties and phytic acid content of the grain were also monitored.

| Cereal grain samples
Local finger millet (brown and dark brown colored) and red-colored sorghum grain varieties were purchased from retail outlet in Thohoyandou, Limpopo province, South Africa. Megazyme K-PHYT assay kit was purchased from Megazyme International Ireland, Bray Business Park, Bray Co. Wicklow, A98 YV29, Ireland. All other chemicals used were of analytical grade.

| Malting of the grain samples
The cereal grains were malted according to the method of Chethan, Sreerama, and Malleshi (2008). Two varieties of finger millet, namely a brown finger millet (BFM) and dark brown finger millet (DBFM), were used in this study while sorghum was used as the control.
Sorghum grain was used as external reference in the study due to the difficulty in obtaining other local varieties of finger millet in the region, and also to validate the technique applied to the test samples. One hundred gram (100 g) portion of the finger millet and sorghum samples was initially soaked in water for 24 hr at 25°C in a growth chamber; the grains were spread on a clean cheese cloth and maintained moist by sprinkling water periodically at intervals of 24 hr. The sprouted grains were kilned for 8 hr at 50°C, after which a characteristic malt aroma was obtained. The kilned grains were milled into fine flour in a stainless steel grinder and stored in polyethylene bags at −20°C until analysis.

| Physicochemical properties of the grain samples
The pH of the milled flour was determined according to AOAC (2000) using a pH meter. Color determination was performed using Lovibond spectrocolorimeter (Model LC 100). Upon calibration of the spectrocolorimeter, the unmalted and malted finger millet and sorghum grains were placed in cuvettes and inserted in the device for color measurement.

| Determination of mineral content of the grain samples
To solubilize the acid-extractable elemental content of the samples, digestion was performed on a MARS microwave digestor, using ultra-pure HNO 3 , or HNO 3 + HCl at elevated temperature and pressure. After a cooling period, the extractant was made up to 50 ml volume with deionized water, then analyzed by ICP-AES and/or ICP-MS for the selected analytes. Major elements were analyzed on a Thermo ICap 6200 ICP-AES. The instrument was calibrated using NIST (National Institute of Standards and Technology, Gaithersburg, MD, USA) traceable standards to quantify selected elements. A NIST-traceable quality control standard of a separate supplier than the main calibration standards were analyzed to verify the accuracy of the calibration before sample analysis. Where sample have undergone a digestion step, the results were corrected for the dilution factor resulting from the digestion procedure. Cobalt and selenium were analyzed on an Agilent 7900 quadrupole ICP-MS. Samples were introduced via a 0.4 ml/min micro-mist nebulizer into a peltiercooled spray chamber at a temperature of 2°C, with a carrier gas flow of 1.05 L/min. The elements were analyzed under He-collision mode to remove polyatomic interferences.

| Phytic acid and total phosphorus content of the grain samples
Phytic acid and total phosphorus content of the unmalted and malted finger millet and sorghum varieties were determined using Megazyme kit (K-PHYT, Megazyme, Bray, Ireland). Accordingly, 1 g of the grain flour was extracted with 20 ml of 0.66 M HCL for 24 hr at room temperature. An aliquot of the extract was centrifuged at 4470 g for 20 min, and the supernatant was neutralized with 0.5 ml 0.75 M NaOH. Neutralized extract was subjected to enzymatic dephosphorylation for free and total phosphorus. For free phosphorus, 0.05 ml of the neutralized extract was mixed with 0.62 ml of distilled water, 0.20 ml 0.02% (w/v) NaN 3 , pH 5.5 sodium acetate buffer, and then vortexed and incubated at 40°C for 10 min. Afterward, 0.02 ml of distilled water, 0.20 ml MgCl 2 , ZnSO 4 , 0.02% NaN 3 , pH 10.4 buffer was added, vortexed, and incubated at 40°C for another 15 min. The reaction was stopped with 0.30 ml (50%) C 2 HCL 3 O 2 . For total phosphorus: 0.05 ml of the neutralized extract was mixed with 0.60 ml of distilled water, 0.20 ml 0.02% w/v NaN 3 , pH 5.5 sodium acetate buffer, and 0.02 ml phytase enzyme. The mixture was vortexed and incubated at 40°C for 10 min before the addition of 0.2 ml MgCl 2 , ZnSO 4 , 0.02% NaN 3 , pH 10.4 buffer and alkaline phosphatase suspension, vortexed, and incubated at 40°C for 15 min. The reaction was stopped by the addition of 50% 0.30 ml C 2 HCL 3 O 2 .
The mixtures were centrifuged at 2860 g for 20 min. Color regent for colorimetric determination of dephosphorylated extract was prepared using 10% w/v ascorbic acid, 1 M) sulfuric acid and 5% w/v ammonium molybdate. One milliliter (1 ml) of dephosphorylated extract (free and total phosphorus) was mixed with 0.5 ml of the color reagent, vortexed, and incubated at 40°C for 1 hr, and absorbance was read at 655 nm. Optical density (OD) for the standard was recorded using the standard solution supplied along with the kit. Total phosphorus and phytic acid contents were calculated as follows: where ΔA phosphorus = Difference between the total phosphorus and free phosphorus

| Statistical analysis
Data obtained were subjected to a one-way ANOVA by Duncan's multiple comparison test using SPSS version 24.0. The mean of the values were considered to be statistically significant at p < 0.05, and Pearson's correlation analysis test was used to analyze the significance of correlations at *p < 0.05 and **p < 0.01.

| Physicochemical properties
The effect of malting period on the pH of the finger millet is presented in Table 1. Malting did not result in a conspicuous decrease in pH of the finger millet varieties and sorghum grain for the entire malting periods. For the grain color properties, the unmalted and malted sorghum grains had higher (p < 0.05) L* values than the corresponding brown and DBFM grains (Figure 1). Lower a* values were observed for unmalted and malted DBFM compared to BFM and sorghum varieties. There was no significant change in yellow color (b*) as a result of malting for DBFM and sorghum grains except for BFM. For chroma (C*) values, changes were observed during malting for DBFM which was lower compared to BFM and sorghum grain. In general, the DBFM had the least color rating compared to BFM and sorghum grains.

| Boron (B)
The amount of B from the unmalted and malted food grains are presented in Figure 2a, and they were in the ranges 1.97-2.30 mg/kg in DBFM, 2.07-2.37 mg/kg in BFM and 1.60-3.23 mg/kg in the sorghum grain. The B content of the sorghum grain was significantly (p < 0.05) higher than the finger millet varieties. Unlike the sorghum grain, the finger millet varieties showed insignificant (p > 0.05) decrease in B content during 24 hr of malting. However, at 48 hr of malting, a significant (p < 0.05) decrease in B content of DBFM was observed, which later increased significantly at 72 and 96 hr to levels closer to that in the unmalted counterpart. For the BFM, no

| Silicon (Si)
The Si content of the unmalted and malted food grains is presented in Figure 2c,

| Calcium (Ca)
The DBFM and BFM varieties were found to contain higher amount of

| Copper (Cu)
Higher amount of Cu was observed in the finger millet varieties compared to the sorghum grain and was in the range 7.07-7.60 mg/kg in DBFM, 6.97-7.77 mg/kg in BFM, and 5.03-5.30 mg/kg in the sorghum grain ( Figure 2e). Reduced (p < 0.05) Cu content was observed during 24 and 72 hr of malting of the millet varieties which later increased, respectively. Increases in the Cu content of BFM were observed at 48 and 96 hr which did not differ significantly from the unmalted grain.
Although 96 hr of malting increased Cu content of DBFM, the amount was significantly lower than the amount observed at 48-hr malt.

| Potassium (K)
Compared to other minerals investigated, the food grains were found to contain higher amount of K which ranged 4,698.80-

| Magnesium (Mg)
The Mg content of the unmalted and malted food grains is shown in Figure 2h, and the amount ranged 2,250-2,374.37 mg/kg in DBFM,

| Phosphorus (P)
The unmalted and malted finger millet varieties and sorghum grain were found to contain high amounts of P which ranged 3,871.

| Sulfur (S)
The S content of the unmalted and malted food grains ranged

| Selenium (Se)
Similar to the Co, lower amount of Se was found in the food grains

| Manganese (Mn)
The amount of Mn present in the unmalted and malted food grains

| Cobalt (Co)
The food grains were found to contain relatively lower amounts of Co (Figure 2o). Malting did not affect the amount of Co in the sorghum grain. However, it resulted in changes in the finger millet varieties and was in the range 0.03-0.04 mg/kg in DBFM and 0.04-0.05 mg/kg in the BFM. An initial decrease (p < 0.05) in Co content was observed at 24 hr for the DBFM which remained constant for the entire malting period. A similar initial decrease was observed for BFM after which there was an increase. Higher amount of Co was recorded at 48 and 72 hr.

| Phytic acid and total phosphorus content
The phytic acid content of the unmalted and malted food grains ranged 0.3363-0.9186 g/100 g in DBFM, 0.5271-1.0680 g/100 g in BFM, and 0.2889-0.6725 g/100 g in the sorghum grain (Table 1).
Malting for 24 hr resulted in a significant (p < 0.05) decrease in the phytic acid content of BFM unlike for DBFM and sorghum grain malt.

| D ISCUSS I ON
The physicochemical properties of the finger millet and sorghum varieties were not strongly affected by the malting process, particularly the pH which became slightly acidic. The changes in the color properties of the finger millet and sorghum grain varieties may have resulted from the distinct color types of the cereal grains, which were slightly modified during the malting.
Finger millet and sorghum grain varieties contain a number of nutritionally essential macro-and microelements and were significantly affected by malting. The amount of calcium, phosphorus, potassium, magnesium, iron, zinc, manganese, copper, and sodium obtained in this study is comparable with the results of Platel et al. (2010), Sripriya et al. (1997), and Shashi, Sharan, Hittalamani, Shankar, and Nagarathna (2007). Higher amounts of calcium (388.9-452.5 mg/100 g), phosphorus (365.8-393.10 mg/100 g), magnesium (211.0-237.4 mg/100 g), zinc (2.68-3.0 mg/100 g), manganese (14.7-19.1 mg/100 g), copper (0.7-0.78 mg/100 g), and sodium (1.9-3.4 mg/100 g), respectively, were observed in our study compared to the value ranges reported by other researchers. The observation could be as a result of the technique and instrument of determination applied, wherein a combination of nitric and hydrochloric acid was used in the extraction of the minerals unlike in other studies where hydrochloric acid is the main extraction solvent. Also, high amount of sulfur (158.4-168.7 mg/100 g) was observed in the finger millet varieties followed by strontium (2.5-3.3 mg/100 g), silicon (2.6-5.4 mg/100 g) and boron (0.2-0.24 mg/100 g) while cobalt (0.003-0.005 mg/100 g) and selenium (0.002-0.005 mg/100 g) were found at very little amounts. The initial 24-hr malting resulted in a decrease in the minerals except for sodium. Loss in sodium content was only observed with malting time. Similar observation has been reported by other researchers (Afify et al., 2011;Krishnan et al., 2012;Malleshi & Desikachar, 1986). As biosynthesis or degradation of minerals is not expected during malting, the loss of the minerals may be due to leaching during soaking and germination stages of the food grains. The change could also may have resulted from loss through utilization of other nutrients by the growing embryo. The increase observed for the sodium content during 24 hr of malting could be due to its highly water soluble nature and single oxidation state which potentiates its early release compared to other minerals.
Relevant changes were noted in the mineral composition of the food grains at 48 up to 96 hr of malting. The effect of malting on phytate-mineral complexes has been well established. Finger millet contain high amount of phytate (417 mg/100 g) compared to other cereals such as sorghum (295 mg/100 g), barley (278 mg/100 g), rice (160 mg/100 g), and maize (414 mg/100 g) (Hemalatha, Platel, & Srinivasan, 2007;Sripriya et al., 1997). Several processing techniques have been shown to reduce the amount of phytate in cereal grains, especially malting, wherein phytase, a phytate-specific enzyme is activated, resulting in the dephosphorylation of inositol phosphates or phytate forms (Luo, Xie, Jin, Wang, & He, 2014). This process often result in phytate complex dissociation and or reconfiguration with principal products being inorganic phosphates, inositol phosphate monomers, divalent and trivalent mineral ions, proteins, and amino acids. Enzymatic hydrolysis of phytate in finger millet during germination was observed by Mbithi-Mwikya, Van Camp, Yiru, and Huyghebaert (2000) wherein phytate values decreased from 0.35 in the raw sample to 0.02 g/100 g after 96 hr of germination. Mamiro et al. (2001) have shown that 48-hr germination reduced 49.2% of the total phytic acid content of finger millet. In another report, germination for 72 hr was found to significantly reduce the phytate content in pearl millet compared to 48 hr germinated (Sehga & Kawatra, 1998).
At 48 hr of malting significant increase in iron, magnesium, phosphorus, sulfur, and zinc were observed for DBFM, whereas in BFM and the sorghum grain, the same was found to increase significantly at 96 hr. At 48 hr, the strontium content of the food grains was higher compared to other malting periods. Also, 48 hr of malting resulted in the increased silicon content of DBFM which reduced the same in BFM and sorghum grain. However, at 72 and 96 hr, respectively, the silicon was found in higher amounts in BFM and sorghum grain. Manganese content was higher at 48 hr of malting for BFM, whereas the same was highest for DBFM at 96 hr. Calcium, copper, and boron were found at a significantly higher amount at 96 hr of malting in the finger millet varieties unlike in the sorghum where higher amounts of the same were observed at 48 hr. Selenium and cobalt content were relatively low of which malting did not result in practical change in their amounts. The increase in the mineral content of the cereal grains observed after 24 hr of malting could be due to an accumulative effect resulting from the dissociation and or reconfiguration of phytate complexes and other mineral binding components of the grain during malting, and as well as the presence of mineral enhancers like ascorbic acids (Krishnan et al., 2012;Mamiro et al., 2001;Sripriya et al., 1997). A varietal difference which is a dependent factor in the degradation of phytate complexes was also found to play a major role in the amount of extractable minerals from the cereal grains. The differences in extractable minerals from the grains, particularly at 48 and 96 hr of malting, could be seen as a function of varietal differences. These results are in contrast with the reports of Platel et al. (2010) and Mamiro et al. (2001) who found no significant effect of malting in the mineral content of finger millet.
The phytic acid content of finger millet and sorghum grain varieties obtained in this study is comparable with the report of Hemalatha et al. (2007) who found 417 mg/100 g (0.417 g/100 g) and 295 mg/100 g (0.295 g/100 g) for finger millet and sorghum grains, respectively. The result is comparable with the report of Mbithi-Mwikya et al. (2000) and Sripriya et al. (1997) who found 0.35 g/100 g and 0.6 g/100 g, respectively. The present result showed that malting had a variable effect on the phytic acid content of the cereal grain varieties as previously described. who showed a steady decrease in phytate content of 0.35 g/z 100 g in the raw sample to 0.02 g/100 g after 96 hr of germination. The discrepancy could arise as a result of the difficulty in estimating accurately the phytic acid content of processed cereal grains which can contain high amounts of other myo-inositol forms (i.e., IP 3 , IP 4 , and IP 5 ) that would co-elute with phytic acid (IP 6 ) and contribute to the total phytic acid content. This is not the case for unprocessed grain where phytic acid comprise at least 97% of the total inositol TA B L E 2 Pearson's correlation coefficients between phytic acid, total phosphorus, power of hydrogen, and minerals of the finger millet and sorghum varieties phosphates, hence the coherence in phytic acid content of the unmalted cereal grain with the report of other researchers.
Phytic acid is present in cereal grains in the form of phytate complexes. During malting, phytases are induced which act on both high and low phytates. Principally, phytase reduces the hexa form of phytic acid (IP6, myo-inositol 1,2,3,4,5,6-hexakisphosphate) into lower forms such as IP5, IP4, IP3, IP2, IP1, and myo-inositol (Agte, Gokhale, & Chiplonkar, 1997). In the process, lower phytic acids IP5, IP4, and IP3 are simultaneously being formed as products from the dissociation and or reconfiguration of other higher inositol phosphates already present like IP5 and IP4 (Qvirist, Carlsson, & Andlid, 2015). This sequence of enzymatic reaction in turn increases the amount of inorganic phosphate and lower phytic acids, which co-elute as total phytic acid content. It is suggested that the differential effect of malting on the phytic acid content of the grains could be as a result of the com- Pearson's correlation coefficients between the minerals, phytic acid, total phosphorus, and pH of the finger millet and sorghum grain malt are presented in Table 2. A significant positive correlation (p < 0.05) was observed between phytic acid, calcium, magnesium, potassium, manganese, and copper for the malting periods particular at 24 and 72 hr, suggesting that changes in phytate content of the grain malt contributed to the release of the minerals. No significant correlation (p > 0.05) was observed between phytic acid, selenium, and boron. This could be as a result of the very minute amounts in which they occur in the finger millet grain. Significant negative correlations between phytic acid, total phosphorus, and zinc were observed. Among the minerals, significant positive and negative correlation coefficients were recorded particularly for cobalt, copper and manganese where negative correlation was observed. Apparently, there are limited studies that have described the interaction between minerals, and phytic acid content of finger millet. Sripriya et al. (1997) observed a decreased phytate/Zn molar ratio from 19.2 to 7.8 for 48hr germinated finger millet, which suggests a negative interaction of phytic acid and zinc availability as observed in the present study.
The pH of plant food plays an important role in availability and/or bioavailability of minerals both in food material and during digestion in the human gut. Under acidic conditions, transition/dissociation of mineral into their ionic state occurs which result in their precipitation under increasing pH, thereby limiting their bioavailability (Skibsted, 2016). In addition, pH affects the hydrolysis of phytate and organic acid complexes which are important for mineral accessibility in cereal grains. The present study showed a significant negative correlation (p < 0.05) between pH and phytate content of the malted grains, thus suggesting that increase in acidity contribute to changes in phytate that affects mineral availability in finger millet.

| CON CLUS ION
Application of ICP-AES/MS reveals that finger millet is a rich source of both macroelements and trace elements in amounts that were not previously reported, and that malting for 72-96 hr positively influenced certain minerals of the grain. Malting for 48 and 96 hr had a better impact on the mineral composition which varied for the grain varieties. The majority of the minerals including magnesium, calcium, phosphorus, iron, zinc, and copper were enhanced at 96 hr of malting for BFM, whereas for DBFM, the reverse was the case except for manganese and strontium. Selenium and cobalt content of the grains were relatively low and were not affected by malting. The changes in phytic acid content of the grain malt suggest that phytate, the principal form of phytic acid present in grains, undergoes dissociation and/ or reconfiguration during malting rather than a degradation of phytic acid. Varietal difference was found to play an important role on how processing method affects the minerals and phytic acid content of the grain. This study provides a rationale for increased utilization of finger millet grain as a functional food ingredient, for the alleviation of mineral deficiencies in children and adult foods.