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In vitro dephytinization and bioavailability of essential minerals in several wheat varieties
Journal of Cereal Science 56 (2012) 741e746
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In vitro dephytinization and bioavailability of essential minerals in several wheat varieties Sharoon Akhter a, Asma Saeed b, Muhammad Irfan a, Kauser Abdullah Malik a, * a b
Department of Biological Sciences, Forman Christian College (A Chartered University), Ferozepur Road, Lahore 54600, Pakistan Food and Biotechnology Research Centre, PCSIR Laboratories Complex, Ferozepur Road, Lahore 54600, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 April 2012 Received in revised form 13 August 2012 Accepted 17 August 2012
Twelve wheat varieties were studied for their phytic acid, calcium, iron, zinc, copper and phosphorus contents, which respectively ranged between 114e166, 25.1e53.5, 3.41e5.55, 0.71e3.00, 0.65e1.32 and 298e314 mg/100 g. The molar ratios as Ca:Phy, Phy:Fe, Phy:Zn, Phy:Cu were found to be 0.14e0.29, 1.96e3.86, 5.11e20.5 and 13.0e23.9, respectively. Significant dephytinization, ranging between 35.3 and 69.3% in different varieties, was achieved on exogenous enzymatic treatment. Enzymatic dephytinization in the in vitro simulated gastrointestinal digestion resulted in significant increase in the bioavailability of essential minerals. The increase in bioavailability of calcium, iron, zinc and copper on exogenous enzymatic dephytinization ranged between, respectively (x-folds): 1.30e1.96, 1.11e1.52, 1.22e1.78 and 1.11e1.73. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Wheat Dephytinization Minerals Bioavailability
1. Introduction Wheat (Triticum aestivum L.) is the world’s second most cultivated cereal crop, after rice (FAO, 2012). It accounts for approximately 30% of the total cereal production worldwide, and being a staple food in most parts of the world, wheat is an important source of principal food constituents, minerals, and energy for billions of people. It is rich in starch along with fibers, also containing non-negligible amounts of proteins, minerals, and vitamins (O’Dell et al., 1972). Wheat also contains a significant amount of phytic acid (Dintzis et al., 1992). Apart from its health benefits, such as antioxidant, anticarcinogen, and in the prevention of coronary diseases (Febles et al., 2002), it is well established that phytic acid also acts as a natural antinutrient substance (Patearroyo and Fernandez-Quintela, 1995). About 60% of minerals and 85% of phytic acid are localized in the aleurone layer of wheat kernel (Itkonen et al., 2012). Milling of wheat to produce refined white flour has been reported to result in a significant loss of dietary fibers, minerals and vitamins, making the process a major cause of their deficiency in the human diet (Frontela et al., 2011).
Abbreviations: ANOVA, analysis of variance; AOAC, Association of Official Analytical Chemists; DM, dialyzate mineral; FAAS, flame atomic absorption spectrophotometer; WHO, World Health Organization. * Corresponding author. Tel.: þ92 42 9923581 8x504; fax: þ92 42 99230703. E-mail addresses: [email protected], [email protected] (K.A. Malik). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcs.2012.08.017
Furthermore, the presence of minerals and phytic acid (myoinositol-1,2,3,4,5,6-hexakisphosphate), together in the aleurone layer, may also affect the bioavailability of minerals due to the possibility of strong chelation between the two (Coudray et al., 2001). Phytic acid is also known to interact with proteins electrostatically, resulting in the formation of insoluble complexes that may reduce the bioavailability and digestibility of minerals and proteins in the gastrointestinal tract due to the low activity of intestinal phytase in human beings (Itkonen et al., 2012). Studies have shown that the human intestine has limited ability to digest phytic acid, whereas phytase is absent in the gut region. The loss, or unavailability, of minerals in the human diet may thus be a contributory cause of malnutrition, impacting severe biological disorders in the human body. Malnutrition, related to calcium, iron, zinc and copper, may lead to several physiological-pathological conditions of a serious nature (Frontela et al., 2011). Another problem is related to the unavailability of phosphorus for absorption, since phytic acid is the major storage compound of cereal grains. It is also significant to note that deficiencies of minerals are commonly observed in the populations of low socioeconomic groups, who normally consume cereals and millets as their staple food (Lakshmi and Sumathi, 1997). Nutritionists have endeavored to increase the bioavailability of minerals, while reducing the level of phytic acid in the human diet. Among the more promising approaches to reduce phytic acid content in cereals is to improve the bioabsorption of minerals through phytase treatment. Phytases are phosphatase enzymes that catalyze the sequential hydrolysis of
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phytic acid to produce less phosphorylated myo-inositol derivatives, mineral complexes, and inorganic phosphates, thus making them nutritionally available (Wyss et al., 1999). Enzymes with phytase-like activities are classified as histidine acid phosphatases (HAPs), purple acid phosphatases (PAPs) and b-propeller phosphatases (Lei et al., 2007). Only HAPs and PAPs have been reported in plants, while the presence of PAP genes have been demonstrated in wheat grains, also confirming that a phytase isogene is responsible for the high phytase activity in transgenic wheat grains (Dionisio et al., 2011). Cloning and characterization of complementary DNAs, encoding a group of enzymes with phytase activity, have been suggested to constitute a significant part of the phytase potential of wheat grains (Dionisio et al., 2007). Pakistan is ranked as the seventh biggest grower of wheat in the world, with an annual production of 24 million tons (FAO, 2012). Therefore, being the major cereal crop, wheat is consumed as a staple food in Pakistan. However, despite the adequate availability and consumption of wheat, several nutritional problems related to micronutrient deficiencies have been reported in the population groups of low socioeconomic status in Pakistan (Ministry of Health, Pakistan, 2001). This ‘hidden hunger’ has manifested itself in the development of several physiological-pathological conditions such as anemia, growth and mental retardation, osteoporosis, and poor immunological responses, due mainly to the subclinical deficiencies of calcium, iron, zinc and copper in children and women. The primary objective of the present study, therefore, was to determine the contents of these essential minerals, and of the antinutrient phytic acid, in twelve Pakistani wheat varieties. The in vitro bioavailability of these minerals in the wheat varieties studied was also determined. This was done by enzymatic dephytinization of wheat and in vitro gastrointestinal digestion, solubilization of the enzymatically hydrolyzed products and dialysis through a semipermeable membrane, and determining the bioavailability of calcium, iron, zinc and copper. The observations were analyzed for the increase of minerals bioavailability as related to the reduction in phytic acid content. 2. Experimental 2.1. Sample collection Grains of twelve different non-transgenic wheat varieties, namely, Faisalabad-2008, Inqlab-91, Lasani-08, Pak-81, Sehar-2006, Shafaq, V-07066, V-07067, V-07071, V-07073, V-07078 and V-07096 were obtained from Dr. Makhdoom Hussain, grown at the Experimental Station of Ayub Agricultural Research Institute, Faisalabad, Pakistan in 2010e2011. Each variety was plotted in 7 rows; 4 m long and 0.2 m apart. The interplant distance in each row was 0.03 m. All varieties were grown under the same environmental conditions, with same application of fertilizers. Wheat grains from each plot were randomly sampled into three subsamples. The grains were milled and sieved through US standards mesh size 40, oven-dried at 100 5 C to constant weights, cooled, and stored in desiccators in air tight bags. 3. Methods 3.1. Total minerals determination Total calcium, iron, zinc and copper contents in the wheat flour samples were determined according to AOAC (2005). Briefly, 5.0 g of each sample was weighed in a pre-weighed crucible, charred, and ashed at 450 5 C in a muffle furnace. The ash was dissolved in 6 N HCl, transferred to 100 ml volumetric flasks, and adjusted to volume with deionized water. The total contents of calcium, iron,
zinc, and copper, present in the wheat flour samples were determined by flame atomic absorption spectrophotometry (FAAS), Unicam 969 (Oxford, UK), using hallow cathode lamps at wavelengths of 422.7 nm, 248.3 nm, 213.9 nm, and 324.8 nm, respectively. The FAAS was calibrated with standard stock solutions of calcium (1000 2 mg/L), iron (1001 2 mg/L), zinc (1001 2 mg/L) and copper (1000 2 mg/L), purchased from Merck (Darmstadt, Germany). For calcium analysis, lanthanum chloride (1%) was added to both standard and the wheat flour samples to avoid interference from phosphorus. 3.2. Total phosphorus determination Total phosphorus in wheat flour samples was determined in accordance with AOAC (2005). Briefly, 1.0 g of the wheat flour samples was ignited at 550 5 C to white or grey ash, followed by the addition of 6 N HCl, warmed, and final volume was made to 100 ml with deionized water in volumetric flasks. Colour was developed using molybdate solution, and absorbance was spectrophotometrically measured at 640 nm (UV-1800, Shimadzu, Japan). Phosphorus content in the wheat flour samples was determined using a standard curve plot. 3.3. Phytic acid determination Phytic acid was extracted in accordance with Latta and Eskin (1980). Briefly, 5.0 g of the wheat flour sample was extracted with 100 ml of 2.4% HCl at room temperature for 1 h. The digest was centrifuged at 5000 rpm for 10 min. The resultant supernatant (5 ml) was passed through Amberlite-IR 400 Cl, previously regenerated with 5% NaOH, followed by the addition of 15 ml of 0.1 M NaCl to remove inorganic phosphorus. Phytic acid was eluted with 15 ml of 0.7 M NaCl. Colour was developed using Wade’s reagent, and the phytic acid content was determined spectrophotometrically at 500 nm (UV-1800, Shimadzu, Japan). The quantity of phytate in the wheat flour samples was calculated from the series of standard solutions of phytic acid through a standard curve plot. Phytase activity was determined in accordance with the procedure of AOAC (1994). A phytase unit (FTU) is defined as the amount of activity that liberates inorganic phosphorus from 1.5 mM Na-phytate solution at a rate of 1 mmol/min at pH 5.5 and 37 C. Phytase activity of the twelve wheat varieties ranged between 4952 and 6521 FTU, which was taken into account for the in vitro dephytinization procedure. 3.4. Dephytinization of wheat flours Wheat flour samples were dephytinized by the exogenous phytase enzyme (Phytezyme) of PAP type from wheat, obtained from Dr. Farooq Latif, B&G Co., Karachi, Pakistan, in accordance with Frontela et al. (2009). Briefly, 35.0 g of the milled and sieved wheat grain flour samples of each variety was transferred to 200 ml of deionized water, followed by the addition of phytase enzyme (Phytezyme) at a concentration of 3.2 units per g of the wheat flour, incubated at pH 5.5 with constant stirring at 55 C for 20 min. The dephytinized wheat flour samples were oven-dried at 120 5 C to constant weights. The samples were ground, sieved through US standards mesh size 40, and packaged in air tight bags. 3.5. In vitro gastrointestinal digestion The in vitro method for the bioavailability assay of calcium, iron, zinc and copper, involving simulated gastrointestinal digestion (double enzyme digestion method), as described by Miller et al. (1981), was adapted for both raw and dephytinized wheat flour
S. Akhter et al. / Journal of Cereal Science 56 (2012) 741e746
samples. Briefly, 5.0 g of the wheat flour samples was homogenized with 20 ml deionized water, adjusted to pH 2 using 6 N HCl, followed by the addition of 1 ml pepsin solution, and incubated at 37 C in a shaking waterbath for 2 h. For intestinal digestion, segments of dialysis tubes containing 25 ml water and the appropriate amount of NaHCO3 to yield pH 7.0 (equivalent to titratable acidity determined previously) were individually placed in different beakers. The beakers were incubated for 30 min at 37 C in a shaking waterbath. Pancreatin bile solution (10 ml) was then added to each digestion beaker, and dialysis was carried out by incubation at 37 C for 2 h in a shaking waterbath. The dialysis tubes were carefully removed, rinsed with deionized water and the dialyzates were collected, which were analyzed for calcium, iron, zinc and copper using FAAS. The dialyzability of minerals was calculated as:
dialyzates
DM 100 % ¼ TM
(A.1)
where DM ¼ dialyzate mineral fraction (mg of mineral/100 g of wheat) and TM ¼ total mineral content (mg of mineral/100 g of wheat). Phytic acid to calcium, iron, zinc and copper mole ratios were calculated by dividing the moles of phytic acid/100 g wheat with the moles of minerals/100 g wheat. 4. Statistical analyses All data on the analysis of phytic acid, and calcium, iron, zinc, copper and phosphorus in the twelve raw and dephytinized wheat flours are presented as mg/100 g dry matter. The bioavailability assays for calcium, iron, zinc and copper in both the raw and dephytinized wheat samples are reported as mg of the mineral availability/100 g of the wheat samples. The calcium:phytate, phytate:iron, phytate:zinc and phytate:copper ratios were calculated to determine the bioavailability of the studied minerals in the presence of phytic acid. The results were analyzed using Duncan’s multiple range test (Steel and Torrie, 1960). A variance analysis (ANOVA) of one factor (dialyzability of calcium, iron, zinc and copper) with respect to raw and dephytinized flour of twelve wheat varieties was also done. A ‘P’ value of less than 0.05 was considered significantly different and is indicated by non-coincidence of the superindices in rows and columns. 5. Results Phytic acid in the studied wheat varieties was the lowest in variety Sehar-2006 and highest in Pak-81 (Table 1). Dephytinization of wheat flour with exogenous phytase resulted in falls of phytic acid contents (from lowest to highest percentage reduction values) to 101 from 156 mg/100 g (35.3% reduction), and to 49.4 from 161 to 51.1 from 166 mg/100 g (69.3/69.2% reduction) in varieties Inqlab-91 and V-07066/Pak-81, respectively. Thus, phytic acid was reduced by about one-half (varieties Faisalabad-08, Sehar2006, Shafaq, Lasani, V-07067, V-07096) to two-third (varieties Pak-81, V-07066, V-07071, V-07073, V-07078) in eleven wheat varieties treated for enzymatic dephytinization, except in the twelfth variety (Inqlab-91) in which the reduction was one-third (Table 1). The contents of calcium, iron, zinc, copper and phosphorus in the twelve wheat varieties are shown in Table 2. Phosphorus contents in the twelve wheat varieties were almost similar, ranging between 298 and 314 mg/100 g. Also calculated were the molar ratios for calcium to phytate, and for phytate to iron, zinc and copper (Table 2).
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Table 1 Effect of enzymatic treatment with exogenous phytase on the phytic acid content of twelve wheat varieties. Wheat variety
Phytic acid content in raw wheat flour (mg/100 g)
Faisalabad-08 Sehar-2006 Pak-81 Shafaq Lasani V-07066 V-07067 V-07071 V-07073 V-07078 V-07096 Inqlab-91
128 114 166 136 135 161 142 151 143 147 126 156
7.51b 18.4c 3.90a 19.0a 19.0a 6.47a 18.9b 14.1a 1.97b 8.20b 0.58c 7.59a
Phytic acid content in dephytinized wheat flour (mg/100 g) 57.9 61.0 51.1 72.7 64.7 49.4 74.4 53.5 55.9 45.9 61.7 101
3.90b 7.93b 2.38c 0.23b 2.69b 4.17c 8.87b 3.19c 2.49c 0.93d 8.32b 4.79a
Reduction in phytic acid (%) 54.8 46.5 69.2 46.5 52.1 69.3 47.6 64.6 60.9 68.8 51.1 35.3
Mean SD values of twelve wheat varieties; n ¼ 3 values; different letters (aed) denote significant differences (P < 0.05) within columns.
The in vitro gastrointestinal bioavailability assays of calcium, iron, zinc and copper in the twelve wheat varieties in raw flours (ve exogenous phytase) and flours dephytinized with exogenous enzyme (þve phytase) are reported in Table 3, from which may be observed that the percentage bioavailability of these minerals increased, respectively, from 16.3 to 31.9 in variety Sehar-2006, from 16.0 to 24.6 in variety V-07071, from 8.36 to 12.7 in variety Lasani, and from 14.9 to 25.0 in variety V-07096. Thus, increase in the bioavailability of different minerals in the twelve wheat varieties upon dephytinization was significant, which on an overall basis for calcium ranged from 21 to 32% in varieties V-07096 and Sehar2006, for iron from 15 to 25% in varieties V-07096 and V-07071, for zinc from 6 to 13% in varieties Pak-81 and Lasani, and for copper from 13 to 23% in varieties Pak-81 and Faisalabad-08. 6. Discussion Phytic acid is found in high concentrations in cereals, reaching 3e6% of the weight of the grain (Febles et al., 2002). Reduction or degradation of phytic acid has been suggested for increasing the bioavailability of minerals bound to it (Adeyeye et al., 2000). This approach is likely to be of significance for those population groups that principally consume cereal-based foods and yet face malnutrition from mineral deficiencies. For this purpose, variety-specific data on phytic acid content has a baseline value. Phytic acid in different Pakistani wheat varieties ranged between 114 and 166 mg/g (Table 1). Other authors have reported phytic acid contents of 209 mg/100 g in Spanish wheat (Frontela et al., 2008), 287 mg/100 g in Nigerian wheat (Adeyeye et al., 2000), 263 mg/ 100 g in Iranian wheat (Gargari et al., 2007), and 117 mg/100 g in Chinese wheat (Ma et al., 2005). However, none of these studies were variety-specific, which is of significance since different varieties have different phytate contents. In broad terms, phytic acid in the Pakistani variety Sehar-2006 was relatively low and comparable to Chinese wheat, whereas all others had higher phytate content than Chinese wheat, but less than Spanish wheat and appreciably less than Iranian and Nigerian wheats. Enzymatic dephytinization in all wheat varieties was significant (Table 1). Since high dephytinization (ranging between 61% and 69%) was observed in varieties Pak-81, V-07078, V-07071, V-07066 and V-07073, these are more suitable for enzymatic reduction of phytic acid. There are no reports in literature on in vitro dephytinization of raw wheat using exogenous phytase in simulated gastrointestinal digestion. Therefore, the present study is the first such report on different varieties of wheat. Earlier studies on
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Table 2 Mean and standard deviation values of calcium, iron, zinc, copper and phosphorus, and their molar ratios to phytic acid content in the raw flours of twelve wheat varieties. Wheat variety
Mineral content (mg/100 g) Ca
Faisalabad-08 Sehar-2006 Pak-81 Shafaq Lasani V-07066 V-07067 V-07071 V-07073 V-07078 V-07096 Inqlab-91
30.6 25.1 39.1 42.6 47.6 51.8 45.8 40.7 30.9 39.2 53.5 33.1
Fe
4.27d 2.79e 3.20c 0.24c 2.27b 4.59a 4.53a 1.94c 3.72d 3.72c 1.66a 1.71d
5.14 4.99 4.88 5.13 4.82 5.55 4.17 4.58 4.79 3.41 3.59 3.48
Molar ratios Zn
0.23b 0.31b 0.17b 0.02b 0.12b 0.01a 0.04c 0.29b 0.17b 0.07d 0.10d 0.04d
1.12 0.87 1.39 0.88 0.71 3.00 1.82 1.07 1.29 0.82 1.24 1.35
Cu
0.03c 0.01d 0.25b 0.04d 0.02f 0.06a 0.09b 0.05c 0.04c 0.02e 0.02c 0.05c
0.82 0.84 0.65 1.04 1.32 1.15 0.94 0.79 1.00 0.65 0.78 0.80
P
0.02c 0.01b 0.05e 0.22b 0.09a 0.02b 0.05b 0.06c 0.02b 0.04e 0.01d 0.01c
298 301 303 305 304 303 306 309 301 306 312 314
0.52h 0.61g 0.53e 0.56d 0.65e 0.60e 0.72d 0.43c 0.52f 0.69d 0.62b 0.57a
Ca:Phy
Phy:Fe
Phy:Zn
Phy:Cu
0.24a 0.28a 0.25a 0.21b 0.19c 0.18c 0.19c 0.23b 0.28a 0.23b 0.14d 0.29a
2.01c 1.96c 2.78b 2.44c 2.56c 2.36c 2.90b 2.91b 2.53c 3.74a 2.94b 3.86a
10.4c 13.2b 11.4c 16.6a 20.5a 5.11e 7.76d 14.6b 10.9c 18.1a 9.97c 11.6b
14.3c 13.2d 23.9a 13.7d 10.7e 13.0d 14.7b 19.1b 13.8d 22.5a 15.5b 19.0b
Mean SD values of twelve wheat varieties; n ¼ 3 values; different letters (aef) denote significant differences (P < 0.05) within columns.
dephytinization have focused on bakery products, composite infant foods, and refined white flour. For example, 50% reduction of phytic acid by fungal phytase in dough for bread-making (Sanz Penella et al., 2008); 88% phytic acid reduction in wheat bran with bakers’ yeast; and significant reduction in phytate content by phytase in composite infant cereals (Frontela et al., 2009). Other studies have reported the bioavailability of different minerals on dephytinization (Frontela et al., 2009, 2011). None of these studies, however, were wheat-specific. Malnutrition related to micronutrient deficiency can create immense socioeconomic problems through a variety of health issues. Furthermore, studies have indicated that human diets with high phytate content significantly decrease the bioavailability of calcium (Kies, 1985), iron (Brune et al., 1992), and zinc (Sandstrom et al., 1987). However, little attention has been paid to generating variety-based data on mineral contents of wheat varieties grown in Pakistan. The range of different minerals determined in the twelve Pakistani varieties was (mg/100 g): calcium between 25.1e53.5; iron between 3.41e5.55; zinc between 0.71e1.82 (3.0 in variety V-07066 was exceptional); copper between 0.65e1.32; and phosphorus between 298e314 (Table 2). These values may be rated as medium to low compared with the Chinese wheat varieties (Zhang et al., 2007). However, minerals in the Pakistani wheat varieties were broadly better, except zinc, than the average range of 43 Chinese wheat cultivars analyzed in another study (Tang et al., 2008), which were reported as (mg/100 g): calcium 33.2 (range 23.7e49.2); iron 2.82 (range 2.16e3.38); zinc 2.86 (range 2.12e3.48); copper 0.69 (range 0.52e0.81); and phosphorus 304.2 (range 227.9e353.5). The low level of zinc (except in variety V-07066) in the Pakistani wheat varieties, as also noted for the Chinese varieties, has been suggested to be probably due to a low level of soil zinc. Some other studies have reported (mg/100 g) iron in Indian wheat flour as 3.39 (Nayak and Nair, 2003); phosphorus in Finish white wheat flour as 116.7 (Itkonen et al., 2012); calcium and zinc in Nigerian wheat as 30.1 and 0.8, respectively (Adeyeye et al., 2000); and zinc and iron in Iranian wheat as 1.40 and 1.82, respectively (Gargari et al., 2007). The inhibitory effect of phytate on mineral availability is well known (Frontela et al., 2011; Gargari et al., 2007). In this connection, phytic acid to minerals ratio, referred to as mineral molar ratio, is important for determining the potential mineral bioavailability, which in general terms indicates higher mineral bioavailability when the molar ratio is low and vice versa. Mineral molar ratios, therefore, have been suggested to be useful for assessing the critical values of the availability of calcium (Abebe et al., 2007), iron and zinc (Gargari et al., 2007). In the present study, calcium:phytate, phytate:iron, phytate:zinc, and phytate:copper molar ratios in
twelve wheat varieties, respectively, ranged between 0.14e0.29; 1.96e3.86; 5.11e20.5; and 10.7e23.9 (Table 2). These overall high mineral molar ratios indicate that the bioavailability of these minerals was restricted. Critical molar ratio for calcium has been reported to be >0.24 (Ma et al., 2007), which in raw materials and fermented dough used in Spanish bakery products was above this value (Frontela et al., 2011). Among the Pakistani wheat varieties, accordingly, only varieties Sehar-2006, Pak-81, V-07073 and Inqlab91 were slightly above this threshold, thus having marginally low calcium bioavailability, while the rest had high to moderate calcium bioavailability. Apparently, phytic acid had little binding attraction for calcium, even though calcium content in the Pakistani wheat varieties was high (Table 2). Critical phytate:iron ratio has been suggested to be >1 (Hallberg et al., 1989), which in infant foods was reported to range from 1.4e3.8 and considered to have the potential to compromise iron bioavailability (Frontela et al., 2009). Since molar ratios for iron in the Pakistani wheat varieties are also within this range, these may be regarded as having the potential for restricted iron bioavailability. It has been suggested that if iron is in the bound form (chelated with phytic acid) its bioavailability is restricted. Another relevant factor is that iron absorption is inhibited when calcium and phytate contents are high, which also was the case in the wheat varieties studied presently (Table 2). Phytate:zinc molar ratio in Iranian wheat flour and the breads made from it, respectively, was 19.36 and 8.91 (Gargari et al., 2007), which are comparable to the values in the studied Pakistani wheat varieties. Phytate:zinc mole ratio of 43.0 in the Nigerian wheat was high, thus having low zinc availability (Adeyeye et al., 2000). Phytate:zinc molar ratios are regarded as an index of zinc bioavailability by International Zinc Nutrition Consultative Group (2004). The cut-off phytate:zinc molar ratios recommended by WHO (1996) are: 15, 5e15 and <5, respectively, for zinc bioavailability as low (10e15%), moderate (30e35%) and high (50e55%). Zinc bioavailability in the Pakistani wheat varieties may be rated as generally moderate, except in variety V-07066 as high and in varieties Shafaq, Lasani and V-07078 as low. Copper is an important cofactor in the redox chemistry of enzymes particularly required for growth and development. However, reports of copper deficiency are less frequent. Nevertheless, its bioavailability for iron absorption is essential, as it plays a significant role during recovery from iron malnutrition (Cámara et al., 2005). Since data on copper bioavailability is scarce (Cámara et al., 2005), no comment on copper bioavailability on the basis of phytate:copper molar ratios of the twelve wheat varieties studied can be made. It has been implied, however, that phytic acid and other well known inhibitors of calcium, iron and zinc bioavailability do not affect bioaccessibility of copper (Cámara et al., 2005).
Cu Zn Fe Ca
Mean SD values of twelve wheat varieties; n ¼ 3 values; different letters (a, b) denote significant variety differences (P < 0.05) between (ve) phytase and (þve) phytase treatments to indicate the effect of dephytinization within rows; different numbers (1e6) denote significant inter-variety differences (P < 0.05) on bioavailability of minerals within columns; within parenthesis are the values of multi-folds increase in bioavailability.
Cu
23.4 18.7 12.9 19.7 15.5 17.8 16.5 18.5 17.2 21.6 25.0 21.0 (1.38) (1.37) (1.30) (1.37) (1.52) (1.32) (1.37) (1.41) (1.22) (1.78) (1.30) (1.23) 1.91b,2 1.54b,2 1.39b,3 1.84b,1 1.42b,1 1.28b,1 1.04b,1 1.84b,2 1.44b,2 1.16b,2 1.63b,2 1.76b,2
Zn
8.28 7.37 6.00 10.7 12.7 10.5 10.2 7.07 6.36 9.29 7.44 8.84 (1.25) (1.40) (1.40) (1.44) (1.44) (1.46) (1.47) (1.52) (1.46) (1.16) (1.11) (1.48) 1.23b,2 1.89b,2 1.73b,2 1.81b,2 1.95b,3 1.13b,2 1.15b,2 1.03b,1 1.43b,2 1.51b,3 1.21b,3 1.71b,2
Fe
18.2 19.6 18.9 19.1 17.2 19.0 19.9 24.6 20.9 15.9 14.5 21.7 (1.84) (1.96) (1.36) (1.62) (1.63) (1.30) (1.53) (1.38) (1.52) (1.55) (1.35) (1.50) 1.05b,2 0.34b,1 1.17b,5 2.15b,2 1.20b,3 0.18b,6 1.12b,3 1.72b,6 1.99b,5 0.87b,3 1.09b,6 1.57b,4
Ca
29.9 31.9 21.8 27.4 24.5 20.4 24.2 22.1 22.1 24.7 20.6 23.9 Faislabad-08 Sehar-2006 Pak-81 Shafaq Lasani V-07066 V-07067 V-07071 V-07073 V-07078 V-07096 Inqlab-91
16.3 16.3 16.0 17.0 15.0 15.6 15.8 16.0 14.5 15.9 15.2 15.9
1.33a 0.14a 0.29a 1.48a 0.79a 0.39a 1.19a 1.77a 0.38a 0.27a 0.56a 1.37a
14.5 14.0 13.4 13.3 12.0 13.0 13.6 16.2 14.4 13.7 13.1 14.7
1.77a,1 1.77a,1 1.87a,1 1.75a,1 2.10a,2 1.77a,2 1.29a,1 1.31a,1 1.66a,1 1.61a,1 1.14a,2 1.89a,1
6.01 5.38 4.62 7.75 8.36 7.96 7.45 5.00 5.21 5.21 5.74 7.19
1.26a,3 1.54a,3 1.37a,4 1.39a,1 1.49a,1 1.89a,1 1.39a,1 1.40a,4 1.43a,3 1.44a,3 1.34a,3 1.31a,2
13.5 13.6 11.7 11.8 10.9 10.9 11.5 16.6 10.7 16.3 14.9 12.2
1.15a,2 2.15a,1 1.54a,3 1.03a,3 1.68a,3 1.18a,3 1.14a,3 1.22a,1 1.17a,3 1.61a,1 1.81a,1 1.93a,2
In vitro bioavailability in dephytinized wheat flour (þve phytase treatment) (%) In vitro bioavailability in raw wheat flours (ve phytase treatment) (%) Wheat variety
Table 3 Effect of dephytinization on phytase enzyme treatment in in vitro simulated gastrointestinal digestion on the bioavailability of calcium, iron, zinc and copper in wheat flours of twelve wheat varieties.
2.63b,2 4.35b,2 1.22b,4 1.28b,2 1.67b,4 1.18b,3 1.35b,4 1.84b,2 1.33b,3 1.06b,2 1.29b,1 1.37b,2
(1.73) (1.37) (1.11) (1.67) (1.42) (1.64) (1.44) (1.12) (1.60) (1.32) (1.68) (1.72)
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745
It has been reported that by removing phytate, bioavailability of minerals in foods can be increased (Frontela et al., 2011). Accordingly, dephytinization of the twelve wheat varieties studied with exogenous phytase, as compared with their raw flours (Table 3), also showed significant (p < 0.05) increase in the bioavailability of minerals. Thus, increase (x-folds) in the bioavailability of calcium, iron, zinc and copper upon dephytinization, respectively, ranged between: 1.3e1.96; 1.11e1.52; 1.22e1.52; and 1.11e1.73. The absorption of iron and zinc is reported to increase 2-folds when the phytate:iron and phytate:zinc molar ratios were reduced in raw flours and infant cereals (Frontela et al., 2008). Similarly, bioavailability of zinc is high when phytate:zinc mole ratio is <5.0 (WHO, 1996). The trend of the mineral bioavailability increase (upper and lower values, respectively) in the twelve wheat varieties was: calcium (varieties Sehar-2006 and V-07066) > copper (varieties Faisalabad08 and Pak-81) > iron (varieties V-07071 and V-07096) ¼ />zinc (varieties Lasani and V-07073). No previous study has reported data on the above aspects of wheat varieties cultivated in Pakistan, which may be useful indicators for wheat variety improvement programmes, soil fortification/amelioration with the deficient minerals, and the efforts to alleviate minerals malnutrition, particularly in the population groups of low socioeconomic status. The data may present opportunities to wheat flour millers and dietitians to develop and recommend variety-based products having appropriate levels of phytic acid and minerals to overcome their specific deficiencies in the population groups suffering from ‘hidden hunger’ related with their bioavailability. 7. Conclusion The data generated in the present study has shown that contents of phytic acid and minerals in different wheat varieties differ significantly. This information provides a baseline for wheat variety improvement programmes, particularly aimed at developing varieties which are low in phytic acid and high in mineral contents. The results can be further useful for variety-specific milling and for developing variety-based products rich in the desired minerals. Significant dephytinization was achieved by exogenous phytase treatment in all the twelve wheat varieties, which resulted in 1.5-folde2-fold bioavailability of minerals in several of these. Enzymatic dephytinization, therefore, has indicated a potential approach to reduce phytates and thus enhance minerals availability in the diet of low socioeconomic population segments whose principal diet is wheat-based. Acknowledgment The work reported here was supported by a competitive research grant by Punjab Agricultural Research Board, Lahore, Pakistan through project # 188 entitled, “Development of wheat by reducing phytates for increasing bioavailability of iron and zinc”. References Abebe, Y., Bogale, A., Hambidge, K.M., Stoecker, B.J., Bailey, K., Gibson, R.S., 2007. Phytate, zinc, iron and calcium content of selected raw and prepared foods consumed in rural Sidama, Southern Ethiopia, and implications for bioavailability. Journal of Food Composition and Analysis 20, 161e168. Adeyeye, E.I., Arogundade, L.A., Akintayo, E.T., Aisida, O.A., Alao, P.A., 2000. Calcium, zinc and phytate interrelationships in some foods of major consumption in Nigeria. Food Chemistry 71, 435e441. AOAC, 1994. Official Methods of Analysis of AOAC International. AOAC International, Washington DC. AOAC, 2005. Official Methods of Analysis of AOAC International, eighteenth ed. AOAC International, Washington DC. Brune, M., Rossander-Hulten, L., Hallberg, L., Gleerup, A., Sandberg, A.S., 1992. Iron adsorption from bread in humans: inhibiting effects of cereal fiber phytate and
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inositol phosphates with different numbers of phosphate groups. Journal of Nutrition 122, 442e449. Cámara, F., Amaro, M.A., Barberá, R., Clemente, G., 2005. Bioaccessibility of minerals in school meals: comparison between dialysis and solubility methods. Food Chemistry 92, 481e489. Coudray, C., Levrat-Verny, M.A., Tressol, J.C., Feillet-Coudray, C., HorcajadaMolteni, N.M., Demigné, C., Rayssiguier, Y., Rémésy, C., 2001. Mineral supplementation of white wheat flour is necessary to maintain adequate mineral status and bone characteristics in rats. Journal of Trace Elements in Medicine and Biology 15, 131e137. Dintzis, F.R., Lehrfeld, J., Nelsen, T.C., Finney, P.L., 1992. Phytate content of soft wheat brans as related to kernel size, cultivar, location, and milling and flour quality parameters. Cereal Chemistry 69, 577e581. Dionisio, G., Holm, P.B., Brinch-Pedersen, H., 2007. Wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) multiple inositol polyphosphate phosphatases (MINPPs) are phytases expressed during grain filling and germination. Plant Biotechnology Journal 5, 325e338. Dionisio, G., Madsen, C.K., Holm, P.B., Welinder, K.G., Jorgensen, M., Stoger, E., Arcalis, E., Brinch-Pedersen, H., 2011. Cloning and characterization of purple acid phosphatase phytases from wheat, barley, maize and rice. Plant Physiology 156, 1087e1100. FAO, 2012. Food and Agriculture Organization. www.faostat.fao.org. Febles, C.I., Arias, A., Hardisson, A., Rodríguez-Alvarez, C., Sierra, A., 2002. Phytic acid level in wheat flours. Journal of Cereal Science 36, 19e23. Frontela, C., García-Alonso, F.J., Ros, G., Martínez, C., 2008. Phytic acid and inositol phosphates in raw flours and infant cereals: the effect of processing. Journal of Food Composition and Analysis 21, 343e350. Frontela, C., Scarino, M.L., Ferruzza, S., Ros, G., Martínez, C., 2009. Effect of dephytinization on bioavailability of iron, calcium and zinc from infant cereals assessed in the Caco-2 cell model. World Journal of Gastroenterology 15, 1977e1984. Frontela, C., Ros, G., Martínez, C., 2011. Phytic acid content “in vitro” iron, calcium and zinc bioavailability in bakery products: the effect of processing. Journal of Cereal Science 54, 173e179. Gargari, B.P., Mahboob, S., Razavieh, S.V., 2007. Content of phytic acid and its mole ratio to zinc in flours and breads consumed in Tabriz, Iran. Food Chemistry 100, 1115e1119. Hallberg, L., Brune, M., Rossander, L., 1989. Iron absorption in man: ascorbic acid and dose-dependent inhibition by phytate. American Journal of Clinical Nutrition 49, 140e144. International Zinc Nutrition Consultative Group (IZiNCG), 2004. Assessment of the risk of zinc deficiency in populations and options for its control. In: Hotz, C., Brown, K.H. (Eds.), Food and Nutrition Bulletin, vol. 25-Suppl. 2, pp. S113eS118. Itkonen, S.T., Ekholm, P.J., Kemi, V.E., Lamberg-Allardt, C.J.E., 2012. Analysis of in vitro digestible phosphorous content in selected processed rye, wheat and barley products. Journal of Food Composition and Analysis 25, 185e189. Kies, C., 1985. Effect of dietary fat and fiber on calcium bioavailability. ACS Symposium Series 12, 175e187.
Lakshmi, R.M., Sumathi, S., 1997. Binding of iron, calcium and zinc by fiber of sorghum and ragi. Food Chemistry 60, 213e217. Latta, M., Eskin, M., 1980. A simple and rapid colorimetric method for phytate determination. Journal of Agricultural and Food Chemistry 28, 1313e1315. Lei, X.G., Porres, J.M., Mullaney, E.J., Brinch-Pedersen, H., 2007. Phytase: source, structure, and application. In Journal of Polaina. In: MacCabe, A.P. (Ed.), Industial Enzymes: Structure, Function and Applications. Springer, Dordrecht, The Netherlands, pp. 505e530. Ma, G., Li, Y., Jin, Y., Zhai, F., Kok, F.J., Yang, X., 2007. Phytate intake and molar ratios of phytate to zinc, iron, and calcium in the diets of people in China. European Journal of Clinical Nutrition 61, 368e374. Ma, G., Jin, Y., Piao, J., Kok, F., Guusje, B., Jacobsen, E., 2005. Phytate, calcium, iron, and zinc contents and their molar ratios in foods commonly consumed in China. Journal of Agricultural and Food Chemistry 53, 10285e10290. Miller, D.D., Schricker, B.R., Rasmussen, R.R., Campen, D.V., 1981. An in vitro method for estimation of iron availability from meals. American Journal of Clinical Nutrition 34, 2248e2256. Ministry of Health, Pakistan, 2001. www.health.gov.pk.202.83.164. Nayak, B., Nair, K.M., 2003. In vitro bioavailability of iron from wheat flour fortified with ascorbic acid, EDTA and sodium hexametaphosphate, with or without iron. Food Chemistry 80, 545e550. O’Dell, B.L., De Borland, A.R., Koirtyohann, S.R., 1972. Distribution of phytate and nutritionally important elements among morphological components of cereal grains. Journal of Agricultural Food Chemistry 20, 718e720. Patearroyo, M.A., Fernandez-Quintela, C.C., 1995. Sustancias antinutritivas en alimentos de origen vegetal. Su significado en la alimentacion humana. Alimentaria 6, 115e120. Sandstrom, B., Almgren, A., Kivisto, B., Cederblad, A., 1987. Zinc absorption in humans from meals based on rye, barley, oatmeal, tritical and whole wheat. Journal of Nutrition 117, 1898e1902. Sanz Penella, J.M., Collar, C., Haros, M., 2008. Effect of wheat bran and enzyme addition on dough functional performance and phytic acid levels in bread. Journal of Cereal Science 48, 715e721. Steel, R.G.D., Torrie, J.H., 1960. Principals and Procedures of Statistics. McGraw, New York. Tang, J., Zou, C., He, Z., Shi, R., Ortiz-Monasterio, I., Qu, Y., Zhang, Y., 2008. Mineral element distributions in milling fractions of Chinese wheats. Journal of Cereal Science 48, 821e828. WHO, 1996. Trace Elements in Human Nutrition and Health. World Health Organization, Geneva, Switzerland, pp. 30e35, 90e91. Wyss, M., Pasamontes, L., Friedlein, A., Remy, R., Tessier, M., Kronenberger, A., Middendorf, A., Lehmann, M., Schnoebelen, L., Rothlisberger, U., Kusznir, E., Wahl, G., Muller, F., Lahm, H.W., Vogel, K., van Loon, A.P.G.M., 1999. Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance. Applied and Environmental Microbiology 65, 359e366. Zhang, Y., Wang, D.S., Zhang, Y., He, Z.H., 2007. Variation of major mineral elements concentration and their relationships in grain of Chinese wheats. Scientia Agricultura Sinica 40, 1871e1876.
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
In vitro dephytinization and bioavailability of essential minerals in several wheat varieties Sharoon Akhter a, Asma Saeed b, Muhammad Irfan a, Kauser Abdullah Malik a, * a b
Department of Biological Sciences, Forman Christian College (A Chartered University), Ferozepur Road, Lahore 54600, Pakistan Food and Biotechnology Research Centre, PCSIR Laboratories Complex, Ferozepur Road, Lahore 54600, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 April 2012 Received in revised form 13 August 2012 Accepted 17 August 2012
Twelve wheat varieties were studied for their phytic acid, calcium, iron, zinc, copper and phosphorus contents, which respectively ranged between 114e166, 25.1e53.5, 3.41e5.55, 0.71e3.00, 0.65e1.32 and 298e314 mg/100 g. The molar ratios as Ca:Phy, Phy:Fe, Phy:Zn, Phy:Cu were found to be 0.14e0.29, 1.96e3.86, 5.11e20.5 and 13.0e23.9, respectively. Significant dephytinization, ranging between 35.3 and 69.3% in different varieties, was achieved on exogenous enzymatic treatment. Enzymatic dephytinization in the in vitro simulated gastrointestinal digestion resulted in significant increase in the bioavailability of essential minerals. The increase in bioavailability of calcium, iron, zinc and copper on exogenous enzymatic dephytinization ranged between, respectively (x-folds): 1.30e1.96, 1.11e1.52, 1.22e1.78 and 1.11e1.73. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Wheat Dephytinization Minerals Bioavailability
1. Introduction Wheat (Triticum aestivum L.) is the world’s second most cultivated cereal crop, after rice (FAO, 2012). It accounts for approximately 30% of the total cereal production worldwide, and being a staple food in most parts of the world, wheat is an important source of principal food constituents, minerals, and energy for billions of people. It is rich in starch along with fibers, also containing non-negligible amounts of proteins, minerals, and vitamins (O’Dell et al., 1972). Wheat also contains a significant amount of phytic acid (Dintzis et al., 1992). Apart from its health benefits, such as antioxidant, anticarcinogen, and in the prevention of coronary diseases (Febles et al., 2002), it is well established that phytic acid also acts as a natural antinutrient substance (Patearroyo and Fernandez-Quintela, 1995). About 60% of minerals and 85% of phytic acid are localized in the aleurone layer of wheat kernel (Itkonen et al., 2012). Milling of wheat to produce refined white flour has been reported to result in a significant loss of dietary fibers, minerals and vitamins, making the process a major cause of their deficiency in the human diet (Frontela et al., 2011).
Abbreviations: ANOVA, analysis of variance; AOAC, Association of Official Analytical Chemists; DM, dialyzate mineral; FAAS, flame atomic absorption spectrophotometer; WHO, World Health Organization. * Corresponding author. Tel.: þ92 42 9923581 8x504; fax: þ92 42 99230703. E-mail addresses: [email protected], [email protected] (K.A. Malik). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcs.2012.08.017
Furthermore, the presence of minerals and phytic acid (myoinositol-1,2,3,4,5,6-hexakisphosphate), together in the aleurone layer, may also affect the bioavailability of minerals due to the possibility of strong chelation between the two (Coudray et al., 2001). Phytic acid is also known to interact with proteins electrostatically, resulting in the formation of insoluble complexes that may reduce the bioavailability and digestibility of minerals and proteins in the gastrointestinal tract due to the low activity of intestinal phytase in human beings (Itkonen et al., 2012). Studies have shown that the human intestine has limited ability to digest phytic acid, whereas phytase is absent in the gut region. The loss, or unavailability, of minerals in the human diet may thus be a contributory cause of malnutrition, impacting severe biological disorders in the human body. Malnutrition, related to calcium, iron, zinc and copper, may lead to several physiological-pathological conditions of a serious nature (Frontela et al., 2011). Another problem is related to the unavailability of phosphorus for absorption, since phytic acid is the major storage compound of cereal grains. It is also significant to note that deficiencies of minerals are commonly observed in the populations of low socioeconomic groups, who normally consume cereals and millets as their staple food (Lakshmi and Sumathi, 1997). Nutritionists have endeavored to increase the bioavailability of minerals, while reducing the level of phytic acid in the human diet. Among the more promising approaches to reduce phytic acid content in cereals is to improve the bioabsorption of minerals through phytase treatment. Phytases are phosphatase enzymes that catalyze the sequential hydrolysis of
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phytic acid to produce less phosphorylated myo-inositol derivatives, mineral complexes, and inorganic phosphates, thus making them nutritionally available (Wyss et al., 1999). Enzymes with phytase-like activities are classified as histidine acid phosphatases (HAPs), purple acid phosphatases (PAPs) and b-propeller phosphatases (Lei et al., 2007). Only HAPs and PAPs have been reported in plants, while the presence of PAP genes have been demonstrated in wheat grains, also confirming that a phytase isogene is responsible for the high phytase activity in transgenic wheat grains (Dionisio et al., 2011). Cloning and characterization of complementary DNAs, encoding a group of enzymes with phytase activity, have been suggested to constitute a significant part of the phytase potential of wheat grains (Dionisio et al., 2007). Pakistan is ranked as the seventh biggest grower of wheat in the world, with an annual production of 24 million tons (FAO, 2012). Therefore, being the major cereal crop, wheat is consumed as a staple food in Pakistan. However, despite the adequate availability and consumption of wheat, several nutritional problems related to micronutrient deficiencies have been reported in the population groups of low socioeconomic status in Pakistan (Ministry of Health, Pakistan, 2001). This ‘hidden hunger’ has manifested itself in the development of several physiological-pathological conditions such as anemia, growth and mental retardation, osteoporosis, and poor immunological responses, due mainly to the subclinical deficiencies of calcium, iron, zinc and copper in children and women. The primary objective of the present study, therefore, was to determine the contents of these essential minerals, and of the antinutrient phytic acid, in twelve Pakistani wheat varieties. The in vitro bioavailability of these minerals in the wheat varieties studied was also determined. This was done by enzymatic dephytinization of wheat and in vitro gastrointestinal digestion, solubilization of the enzymatically hydrolyzed products and dialysis through a semipermeable membrane, and determining the bioavailability of calcium, iron, zinc and copper. The observations were analyzed for the increase of minerals bioavailability as related to the reduction in phytic acid content. 2. Experimental 2.1. Sample collection Grains of twelve different non-transgenic wheat varieties, namely, Faisalabad-2008, Inqlab-91, Lasani-08, Pak-81, Sehar-2006, Shafaq, V-07066, V-07067, V-07071, V-07073, V-07078 and V-07096 were obtained from Dr. Makhdoom Hussain, grown at the Experimental Station of Ayub Agricultural Research Institute, Faisalabad, Pakistan in 2010e2011. Each variety was plotted in 7 rows; 4 m long and 0.2 m apart. The interplant distance in each row was 0.03 m. All varieties were grown under the same environmental conditions, with same application of fertilizers. Wheat grains from each plot were randomly sampled into three subsamples. The grains were milled and sieved through US standards mesh size 40, oven-dried at 100 5 C to constant weights, cooled, and stored in desiccators in air tight bags. 3. Methods 3.1. Total minerals determination Total calcium, iron, zinc and copper contents in the wheat flour samples were determined according to AOAC (2005). Briefly, 5.0 g of each sample was weighed in a pre-weighed crucible, charred, and ashed at 450 5 C in a muffle furnace. The ash was dissolved in 6 N HCl, transferred to 100 ml volumetric flasks, and adjusted to volume with deionized water. The total contents of calcium, iron,
zinc, and copper, present in the wheat flour samples were determined by flame atomic absorption spectrophotometry (FAAS), Unicam 969 (Oxford, UK), using hallow cathode lamps at wavelengths of 422.7 nm, 248.3 nm, 213.9 nm, and 324.8 nm, respectively. The FAAS was calibrated with standard stock solutions of calcium (1000 2 mg/L), iron (1001 2 mg/L), zinc (1001 2 mg/L) and copper (1000 2 mg/L), purchased from Merck (Darmstadt, Germany). For calcium analysis, lanthanum chloride (1%) was added to both standard and the wheat flour samples to avoid interference from phosphorus. 3.2. Total phosphorus determination Total phosphorus in wheat flour samples was determined in accordance with AOAC (2005). Briefly, 1.0 g of the wheat flour samples was ignited at 550 5 C to white or grey ash, followed by the addition of 6 N HCl, warmed, and final volume was made to 100 ml with deionized water in volumetric flasks. Colour was developed using molybdate solution, and absorbance was spectrophotometrically measured at 640 nm (UV-1800, Shimadzu, Japan). Phosphorus content in the wheat flour samples was determined using a standard curve plot. 3.3. Phytic acid determination Phytic acid was extracted in accordance with Latta and Eskin (1980). Briefly, 5.0 g of the wheat flour sample was extracted with 100 ml of 2.4% HCl at room temperature for 1 h. The digest was centrifuged at 5000 rpm for 10 min. The resultant supernatant (5 ml) was passed through Amberlite-IR 400 Cl, previously regenerated with 5% NaOH, followed by the addition of 15 ml of 0.1 M NaCl to remove inorganic phosphorus. Phytic acid was eluted with 15 ml of 0.7 M NaCl. Colour was developed using Wade’s reagent, and the phytic acid content was determined spectrophotometrically at 500 nm (UV-1800, Shimadzu, Japan). The quantity of phytate in the wheat flour samples was calculated from the series of standard solutions of phytic acid through a standard curve plot. Phytase activity was determined in accordance with the procedure of AOAC (1994). A phytase unit (FTU) is defined as the amount of activity that liberates inorganic phosphorus from 1.5 mM Na-phytate solution at a rate of 1 mmol/min at pH 5.5 and 37 C. Phytase activity of the twelve wheat varieties ranged between 4952 and 6521 FTU, which was taken into account for the in vitro dephytinization procedure. 3.4. Dephytinization of wheat flours Wheat flour samples were dephytinized by the exogenous phytase enzyme (Phytezyme) of PAP type from wheat, obtained from Dr. Farooq Latif, B&G Co., Karachi, Pakistan, in accordance with Frontela et al. (2009). Briefly, 35.0 g of the milled and sieved wheat grain flour samples of each variety was transferred to 200 ml of deionized water, followed by the addition of phytase enzyme (Phytezyme) at a concentration of 3.2 units per g of the wheat flour, incubated at pH 5.5 with constant stirring at 55 C for 20 min. The dephytinized wheat flour samples were oven-dried at 120 5 C to constant weights. The samples were ground, sieved through US standards mesh size 40, and packaged in air tight bags. 3.5. In vitro gastrointestinal digestion The in vitro method for the bioavailability assay of calcium, iron, zinc and copper, involving simulated gastrointestinal digestion (double enzyme digestion method), as described by Miller et al. (1981), was adapted for both raw and dephytinized wheat flour
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samples. Briefly, 5.0 g of the wheat flour samples was homogenized with 20 ml deionized water, adjusted to pH 2 using 6 N HCl, followed by the addition of 1 ml pepsin solution, and incubated at 37 C in a shaking waterbath for 2 h. For intestinal digestion, segments of dialysis tubes containing 25 ml water and the appropriate amount of NaHCO3 to yield pH 7.0 (equivalent to titratable acidity determined previously) were individually placed in different beakers. The beakers were incubated for 30 min at 37 C in a shaking waterbath. Pancreatin bile solution (10 ml) was then added to each digestion beaker, and dialysis was carried out by incubation at 37 C for 2 h in a shaking waterbath. The dialysis tubes were carefully removed, rinsed with deionized water and the dialyzates were collected, which were analyzed for calcium, iron, zinc and copper using FAAS. The dialyzability of minerals was calculated as:
dialyzates
DM 100 % ¼ TM
(A.1)
where DM ¼ dialyzate mineral fraction (mg of mineral/100 g of wheat) and TM ¼ total mineral content (mg of mineral/100 g of wheat). Phytic acid to calcium, iron, zinc and copper mole ratios were calculated by dividing the moles of phytic acid/100 g wheat with the moles of minerals/100 g wheat. 4. Statistical analyses All data on the analysis of phytic acid, and calcium, iron, zinc, copper and phosphorus in the twelve raw and dephytinized wheat flours are presented as mg/100 g dry matter. The bioavailability assays for calcium, iron, zinc and copper in both the raw and dephytinized wheat samples are reported as mg of the mineral availability/100 g of the wheat samples. The calcium:phytate, phytate:iron, phytate:zinc and phytate:copper ratios were calculated to determine the bioavailability of the studied minerals in the presence of phytic acid. The results were analyzed using Duncan’s multiple range test (Steel and Torrie, 1960). A variance analysis (ANOVA) of one factor (dialyzability of calcium, iron, zinc and copper) with respect to raw and dephytinized flour of twelve wheat varieties was also done. A ‘P’ value of less than 0.05 was considered significantly different and is indicated by non-coincidence of the superindices in rows and columns. 5. Results Phytic acid in the studied wheat varieties was the lowest in variety Sehar-2006 and highest in Pak-81 (Table 1). Dephytinization of wheat flour with exogenous phytase resulted in falls of phytic acid contents (from lowest to highest percentage reduction values) to 101 from 156 mg/100 g (35.3% reduction), and to 49.4 from 161 to 51.1 from 166 mg/100 g (69.3/69.2% reduction) in varieties Inqlab-91 and V-07066/Pak-81, respectively. Thus, phytic acid was reduced by about one-half (varieties Faisalabad-08, Sehar2006, Shafaq, Lasani, V-07067, V-07096) to two-third (varieties Pak-81, V-07066, V-07071, V-07073, V-07078) in eleven wheat varieties treated for enzymatic dephytinization, except in the twelfth variety (Inqlab-91) in which the reduction was one-third (Table 1). The contents of calcium, iron, zinc, copper and phosphorus in the twelve wheat varieties are shown in Table 2. Phosphorus contents in the twelve wheat varieties were almost similar, ranging between 298 and 314 mg/100 g. Also calculated were the molar ratios for calcium to phytate, and for phytate to iron, zinc and copper (Table 2).
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Table 1 Effect of enzymatic treatment with exogenous phytase on the phytic acid content of twelve wheat varieties. Wheat variety
Phytic acid content in raw wheat flour (mg/100 g)
Faisalabad-08 Sehar-2006 Pak-81 Shafaq Lasani V-07066 V-07067 V-07071 V-07073 V-07078 V-07096 Inqlab-91
128 114 166 136 135 161 142 151 143 147 126 156
7.51b 18.4c 3.90a 19.0a 19.0a 6.47a 18.9b 14.1a 1.97b 8.20b 0.58c 7.59a
Phytic acid content in dephytinized wheat flour (mg/100 g) 57.9 61.0 51.1 72.7 64.7 49.4 74.4 53.5 55.9 45.9 61.7 101
3.90b 7.93b 2.38c 0.23b 2.69b 4.17c 8.87b 3.19c 2.49c 0.93d 8.32b 4.79a
Reduction in phytic acid (%) 54.8 46.5 69.2 46.5 52.1 69.3 47.6 64.6 60.9 68.8 51.1 35.3
Mean SD values of twelve wheat varieties; n ¼ 3 values; different letters (aed) denote significant differences (P < 0.05) within columns.
The in vitro gastrointestinal bioavailability assays of calcium, iron, zinc and copper in the twelve wheat varieties in raw flours (ve exogenous phytase) and flours dephytinized with exogenous enzyme (þve phytase) are reported in Table 3, from which may be observed that the percentage bioavailability of these minerals increased, respectively, from 16.3 to 31.9 in variety Sehar-2006, from 16.0 to 24.6 in variety V-07071, from 8.36 to 12.7 in variety Lasani, and from 14.9 to 25.0 in variety V-07096. Thus, increase in the bioavailability of different minerals in the twelve wheat varieties upon dephytinization was significant, which on an overall basis for calcium ranged from 21 to 32% in varieties V-07096 and Sehar2006, for iron from 15 to 25% in varieties V-07096 and V-07071, for zinc from 6 to 13% in varieties Pak-81 and Lasani, and for copper from 13 to 23% in varieties Pak-81 and Faisalabad-08. 6. Discussion Phytic acid is found in high concentrations in cereals, reaching 3e6% of the weight of the grain (Febles et al., 2002). Reduction or degradation of phytic acid has been suggested for increasing the bioavailability of minerals bound to it (Adeyeye et al., 2000). This approach is likely to be of significance for those population groups that principally consume cereal-based foods and yet face malnutrition from mineral deficiencies. For this purpose, variety-specific data on phytic acid content has a baseline value. Phytic acid in different Pakistani wheat varieties ranged between 114 and 166 mg/g (Table 1). Other authors have reported phytic acid contents of 209 mg/100 g in Spanish wheat (Frontela et al., 2008), 287 mg/100 g in Nigerian wheat (Adeyeye et al., 2000), 263 mg/ 100 g in Iranian wheat (Gargari et al., 2007), and 117 mg/100 g in Chinese wheat (Ma et al., 2005). However, none of these studies were variety-specific, which is of significance since different varieties have different phytate contents. In broad terms, phytic acid in the Pakistani variety Sehar-2006 was relatively low and comparable to Chinese wheat, whereas all others had higher phytate content than Chinese wheat, but less than Spanish wheat and appreciably less than Iranian and Nigerian wheats. Enzymatic dephytinization in all wheat varieties was significant (Table 1). Since high dephytinization (ranging between 61% and 69%) was observed in varieties Pak-81, V-07078, V-07071, V-07066 and V-07073, these are more suitable for enzymatic reduction of phytic acid. There are no reports in literature on in vitro dephytinization of raw wheat using exogenous phytase in simulated gastrointestinal digestion. Therefore, the present study is the first such report on different varieties of wheat. Earlier studies on
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Table 2 Mean and standard deviation values of calcium, iron, zinc, copper and phosphorus, and their molar ratios to phytic acid content in the raw flours of twelve wheat varieties. Wheat variety
Mineral content (mg/100 g) Ca
Faisalabad-08 Sehar-2006 Pak-81 Shafaq Lasani V-07066 V-07067 V-07071 V-07073 V-07078 V-07096 Inqlab-91
30.6 25.1 39.1 42.6 47.6 51.8 45.8 40.7 30.9 39.2 53.5 33.1
Fe
4.27d 2.79e 3.20c 0.24c 2.27b 4.59a 4.53a 1.94c 3.72d 3.72c 1.66a 1.71d
5.14 4.99 4.88 5.13 4.82 5.55 4.17 4.58 4.79 3.41 3.59 3.48
Molar ratios Zn
0.23b 0.31b 0.17b 0.02b 0.12b 0.01a 0.04c 0.29b 0.17b 0.07d 0.10d 0.04d
1.12 0.87 1.39 0.88 0.71 3.00 1.82 1.07 1.29 0.82 1.24 1.35
Cu
0.03c 0.01d 0.25b 0.04d 0.02f 0.06a 0.09b 0.05c 0.04c 0.02e 0.02c 0.05c
0.82 0.84 0.65 1.04 1.32 1.15 0.94 0.79 1.00 0.65 0.78 0.80
P
0.02c 0.01b 0.05e 0.22b 0.09a 0.02b 0.05b 0.06c 0.02b 0.04e 0.01d 0.01c
298 301 303 305 304 303 306 309 301 306 312 314
0.52h 0.61g 0.53e 0.56d 0.65e 0.60e 0.72d 0.43c 0.52f 0.69d 0.62b 0.57a
Ca:Phy
Phy:Fe
Phy:Zn
Phy:Cu
0.24a 0.28a 0.25a 0.21b 0.19c 0.18c 0.19c 0.23b 0.28a 0.23b 0.14d 0.29a
2.01c 1.96c 2.78b 2.44c 2.56c 2.36c 2.90b 2.91b 2.53c 3.74a 2.94b 3.86a
10.4c 13.2b 11.4c 16.6a 20.5a 5.11e 7.76d 14.6b 10.9c 18.1a 9.97c 11.6b
14.3c 13.2d 23.9a 13.7d 10.7e 13.0d 14.7b 19.1b 13.8d 22.5a 15.5b 19.0b
Mean SD values of twelve wheat varieties; n ¼ 3 values; different letters (aef) denote significant differences (P < 0.05) within columns.
dephytinization have focused on bakery products, composite infant foods, and refined white flour. For example, 50% reduction of phytic acid by fungal phytase in dough for bread-making (Sanz Penella et al., 2008); 88% phytic acid reduction in wheat bran with bakers’ yeast; and significant reduction in phytate content by phytase in composite infant cereals (Frontela et al., 2009). Other studies have reported the bioavailability of different minerals on dephytinization (Frontela et al., 2009, 2011). None of these studies, however, were wheat-specific. Malnutrition related to micronutrient deficiency can create immense socioeconomic problems through a variety of health issues. Furthermore, studies have indicated that human diets with high phytate content significantly decrease the bioavailability of calcium (Kies, 1985), iron (Brune et al., 1992), and zinc (Sandstrom et al., 1987). However, little attention has been paid to generating variety-based data on mineral contents of wheat varieties grown in Pakistan. The range of different minerals determined in the twelve Pakistani varieties was (mg/100 g): calcium between 25.1e53.5; iron between 3.41e5.55; zinc between 0.71e1.82 (3.0 in variety V-07066 was exceptional); copper between 0.65e1.32; and phosphorus between 298e314 (Table 2). These values may be rated as medium to low compared with the Chinese wheat varieties (Zhang et al., 2007). However, minerals in the Pakistani wheat varieties were broadly better, except zinc, than the average range of 43 Chinese wheat cultivars analyzed in another study (Tang et al., 2008), which were reported as (mg/100 g): calcium 33.2 (range 23.7e49.2); iron 2.82 (range 2.16e3.38); zinc 2.86 (range 2.12e3.48); copper 0.69 (range 0.52e0.81); and phosphorus 304.2 (range 227.9e353.5). The low level of zinc (except in variety V-07066) in the Pakistani wheat varieties, as also noted for the Chinese varieties, has been suggested to be probably due to a low level of soil zinc. Some other studies have reported (mg/100 g) iron in Indian wheat flour as 3.39 (Nayak and Nair, 2003); phosphorus in Finish white wheat flour as 116.7 (Itkonen et al., 2012); calcium and zinc in Nigerian wheat as 30.1 and 0.8, respectively (Adeyeye et al., 2000); and zinc and iron in Iranian wheat as 1.40 and 1.82, respectively (Gargari et al., 2007). The inhibitory effect of phytate on mineral availability is well known (Frontela et al., 2011; Gargari et al., 2007). In this connection, phytic acid to minerals ratio, referred to as mineral molar ratio, is important for determining the potential mineral bioavailability, which in general terms indicates higher mineral bioavailability when the molar ratio is low and vice versa. Mineral molar ratios, therefore, have been suggested to be useful for assessing the critical values of the availability of calcium (Abebe et al., 2007), iron and zinc (Gargari et al., 2007). In the present study, calcium:phytate, phytate:iron, phytate:zinc, and phytate:copper molar ratios in
twelve wheat varieties, respectively, ranged between 0.14e0.29; 1.96e3.86; 5.11e20.5; and 10.7e23.9 (Table 2). These overall high mineral molar ratios indicate that the bioavailability of these minerals was restricted. Critical molar ratio for calcium has been reported to be >0.24 (Ma et al., 2007), which in raw materials and fermented dough used in Spanish bakery products was above this value (Frontela et al., 2011). Among the Pakistani wheat varieties, accordingly, only varieties Sehar-2006, Pak-81, V-07073 and Inqlab91 were slightly above this threshold, thus having marginally low calcium bioavailability, while the rest had high to moderate calcium bioavailability. Apparently, phytic acid had little binding attraction for calcium, even though calcium content in the Pakistani wheat varieties was high (Table 2). Critical phytate:iron ratio has been suggested to be >1 (Hallberg et al., 1989), which in infant foods was reported to range from 1.4e3.8 and considered to have the potential to compromise iron bioavailability (Frontela et al., 2009). Since molar ratios for iron in the Pakistani wheat varieties are also within this range, these may be regarded as having the potential for restricted iron bioavailability. It has been suggested that if iron is in the bound form (chelated with phytic acid) its bioavailability is restricted. Another relevant factor is that iron absorption is inhibited when calcium and phytate contents are high, which also was the case in the wheat varieties studied presently (Table 2). Phytate:zinc molar ratio in Iranian wheat flour and the breads made from it, respectively, was 19.36 and 8.91 (Gargari et al., 2007), which are comparable to the values in the studied Pakistani wheat varieties. Phytate:zinc mole ratio of 43.0 in the Nigerian wheat was high, thus having low zinc availability (Adeyeye et al., 2000). Phytate:zinc molar ratios are regarded as an index of zinc bioavailability by International Zinc Nutrition Consultative Group (2004). The cut-off phytate:zinc molar ratios recommended by WHO (1996) are: 15, 5e15 and <5, respectively, for zinc bioavailability as low (10e15%), moderate (30e35%) and high (50e55%). Zinc bioavailability in the Pakistani wheat varieties may be rated as generally moderate, except in variety V-07066 as high and in varieties Shafaq, Lasani and V-07078 as low. Copper is an important cofactor in the redox chemistry of enzymes particularly required for growth and development. However, reports of copper deficiency are less frequent. Nevertheless, its bioavailability for iron absorption is essential, as it plays a significant role during recovery from iron malnutrition (Cámara et al., 2005). Since data on copper bioavailability is scarce (Cámara et al., 2005), no comment on copper bioavailability on the basis of phytate:copper molar ratios of the twelve wheat varieties studied can be made. It has been implied, however, that phytic acid and other well known inhibitors of calcium, iron and zinc bioavailability do not affect bioaccessibility of copper (Cámara et al., 2005).
Cu Zn Fe Ca
Mean SD values of twelve wheat varieties; n ¼ 3 values; different letters (a, b) denote significant variety differences (P < 0.05) between (ve) phytase and (þve) phytase treatments to indicate the effect of dephytinization within rows; different numbers (1e6) denote significant inter-variety differences (P < 0.05) on bioavailability of minerals within columns; within parenthesis are the values of multi-folds increase in bioavailability.
Cu
23.4 18.7 12.9 19.7 15.5 17.8 16.5 18.5 17.2 21.6 25.0 21.0 (1.38) (1.37) (1.30) (1.37) (1.52) (1.32) (1.37) (1.41) (1.22) (1.78) (1.30) (1.23) 1.91b,2 1.54b,2 1.39b,3 1.84b,1 1.42b,1 1.28b,1 1.04b,1 1.84b,2 1.44b,2 1.16b,2 1.63b,2 1.76b,2
Zn
8.28 7.37 6.00 10.7 12.7 10.5 10.2 7.07 6.36 9.29 7.44 8.84 (1.25) (1.40) (1.40) (1.44) (1.44) (1.46) (1.47) (1.52) (1.46) (1.16) (1.11) (1.48) 1.23b,2 1.89b,2 1.73b,2 1.81b,2 1.95b,3 1.13b,2 1.15b,2 1.03b,1 1.43b,2 1.51b,3 1.21b,3 1.71b,2
Fe
18.2 19.6 18.9 19.1 17.2 19.0 19.9 24.6 20.9 15.9 14.5 21.7 (1.84) (1.96) (1.36) (1.62) (1.63) (1.30) (1.53) (1.38) (1.52) (1.55) (1.35) (1.50) 1.05b,2 0.34b,1 1.17b,5 2.15b,2 1.20b,3 0.18b,6 1.12b,3 1.72b,6 1.99b,5 0.87b,3 1.09b,6 1.57b,4
Ca
29.9 31.9 21.8 27.4 24.5 20.4 24.2 22.1 22.1 24.7 20.6 23.9 Faislabad-08 Sehar-2006 Pak-81 Shafaq Lasani V-07066 V-07067 V-07071 V-07073 V-07078 V-07096 Inqlab-91
16.3 16.3 16.0 17.0 15.0 15.6 15.8 16.0 14.5 15.9 15.2 15.9
1.33a 0.14a 0.29a 1.48a 0.79a 0.39a 1.19a 1.77a 0.38a 0.27a 0.56a 1.37a
14.5 14.0 13.4 13.3 12.0 13.0 13.6 16.2 14.4 13.7 13.1 14.7
1.77a,1 1.77a,1 1.87a,1 1.75a,1 2.10a,2 1.77a,2 1.29a,1 1.31a,1 1.66a,1 1.61a,1 1.14a,2 1.89a,1
6.01 5.38 4.62 7.75 8.36 7.96 7.45 5.00 5.21 5.21 5.74 7.19
1.26a,3 1.54a,3 1.37a,4 1.39a,1 1.49a,1 1.89a,1 1.39a,1 1.40a,4 1.43a,3 1.44a,3 1.34a,3 1.31a,2
13.5 13.6 11.7 11.8 10.9 10.9 11.5 16.6 10.7 16.3 14.9 12.2
1.15a,2 2.15a,1 1.54a,3 1.03a,3 1.68a,3 1.18a,3 1.14a,3 1.22a,1 1.17a,3 1.61a,1 1.81a,1 1.93a,2
In vitro bioavailability in dephytinized wheat flour (þve phytase treatment) (%) In vitro bioavailability in raw wheat flours (ve phytase treatment) (%) Wheat variety
Table 3 Effect of dephytinization on phytase enzyme treatment in in vitro simulated gastrointestinal digestion on the bioavailability of calcium, iron, zinc and copper in wheat flours of twelve wheat varieties.
2.63b,2 4.35b,2 1.22b,4 1.28b,2 1.67b,4 1.18b,3 1.35b,4 1.84b,2 1.33b,3 1.06b,2 1.29b,1 1.37b,2
(1.73) (1.37) (1.11) (1.67) (1.42) (1.64) (1.44) (1.12) (1.60) (1.32) (1.68) (1.72)
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It has been reported that by removing phytate, bioavailability of minerals in foods can be increased (Frontela et al., 2011). Accordingly, dephytinization of the twelve wheat varieties studied with exogenous phytase, as compared with their raw flours (Table 3), also showed significant (p < 0.05) increase in the bioavailability of minerals. Thus, increase (x-folds) in the bioavailability of calcium, iron, zinc and copper upon dephytinization, respectively, ranged between: 1.3e1.96; 1.11e1.52; 1.22e1.52; and 1.11e1.73. The absorption of iron and zinc is reported to increase 2-folds when the phytate:iron and phytate:zinc molar ratios were reduced in raw flours and infant cereals (Frontela et al., 2008). Similarly, bioavailability of zinc is high when phytate:zinc mole ratio is <5.0 (WHO, 1996). The trend of the mineral bioavailability increase (upper and lower values, respectively) in the twelve wheat varieties was: calcium (varieties Sehar-2006 and V-07066) > copper (varieties Faisalabad08 and Pak-81) > iron (varieties V-07071 and V-07096) ¼ />zinc (varieties Lasani and V-07073). No previous study has reported data on the above aspects of wheat varieties cultivated in Pakistan, which may be useful indicators for wheat variety improvement programmes, soil fortification/amelioration with the deficient minerals, and the efforts to alleviate minerals malnutrition, particularly in the population groups of low socioeconomic status. The data may present opportunities to wheat flour millers and dietitians to develop and recommend variety-based products having appropriate levels of phytic acid and minerals to overcome their specific deficiencies in the population groups suffering from ‘hidden hunger’ related with their bioavailability. 7. Conclusion The data generated in the present study has shown that contents of phytic acid and minerals in different wheat varieties differ significantly. This information provides a baseline for wheat variety improvement programmes, particularly aimed at developing varieties which are low in phytic acid and high in mineral contents. The results can be further useful for variety-specific milling and for developing variety-based products rich in the desired minerals. Significant dephytinization was achieved by exogenous phytase treatment in all the twelve wheat varieties, which resulted in 1.5-folde2-fold bioavailability of minerals in several of these. Enzymatic dephytinization, therefore, has indicated a potential approach to reduce phytates and thus enhance minerals availability in the diet of low socioeconomic population segments whose principal diet is wheat-based. Acknowledgment The work reported here was supported by a competitive research grant by Punjab Agricultural Research Board, Lahore, Pakistan through project # 188 entitled, “Development of wheat by reducing phytates for increasing bioavailability of iron and zinc”. References Abebe, Y., Bogale, A., Hambidge, K.M., Stoecker, B.J., Bailey, K., Gibson, R.S., 2007. Phytate, zinc, iron and calcium content of selected raw and prepared foods consumed in rural Sidama, Southern Ethiopia, and implications for bioavailability. Journal of Food Composition and Analysis 20, 161e168. Adeyeye, E.I., Arogundade, L.A., Akintayo, E.T., Aisida, O.A., Alao, P.A., 2000. Calcium, zinc and phytate interrelationships in some foods of major consumption in Nigeria. Food Chemistry 71, 435e441. AOAC, 1994. Official Methods of Analysis of AOAC International. AOAC International, Washington DC. AOAC, 2005. Official Methods of Analysis of AOAC International, eighteenth ed. AOAC International, Washington DC. Brune, M., Rossander-Hulten, L., Hallberg, L., Gleerup, A., Sandberg, A.S., 1992. Iron adsorption from bread in humans: inhibiting effects of cereal fiber phytate and
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