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Effect of phytic acid on iron bioavailability in fortified infant cereals Sedef Nehir El, Sibel Karakaya and S ebnem S imsek
Effect of phytic acid in cereals 485
Food Engineering Department, Engineering Faculty, Ege University, I_zmir, Turkey Abstract Purpose – Iron deficiency is an important nutritional problem that affects approximately 20 percent of world’s population and especially infants. The aim of this paper is to determine the iron bioavailability by using in vitro method in commercial infant cereals. Design/methodology/approach – The ferrous iron dialyzability relative to total iron and phytic acid contents of six commercial infant cereals commonly consumed in Turkey were analyzed. Findings – Dialyzable ferrous iron was determined in samples 4, 5, and 6 as 2.51 0.38, 4.12 1.52, and 0.50 0.08 percent, respectively ( p < 0.05). Phytic acid contents of the samples ranged from 118 to 161 mg/100 g. For all the samples calculated phytate:iron molar ratios were equal to or higher than 1 (ranged from 1.0 to 9.89). Originality/value – The phytate:iron molar ratio was not found as the major inhibitory factor on iron bioavailability. Other possible factors such as type of iron fortificant and possible interactions of iron with other ingredients in the formula can affect iron bioavailability. Therefore, at the formulation step amounts of all ingredients and their proportions to each other should be considered to reach optimum iron bioavailability. Keywords Cereal foods, Infants, Minerals, Personal health Paper type Research paper
Introduction Iron (Fe) deficiency is an important nutritional problem that affects approximately 20 percent of world’s population. The main risk groups for the deficiency both in Turkey and the world are infants, children, pregnant women, and women at childbearing age. In Turkey the rate of anemia in women is estimated as just over 30 percent and in young children just over 20 percent (Pekcan, 2001; Stoltzfus et al., 2009). Especially, effects of iron deficiency on normal neural development and potential irreversible nature of this alteration in infants need to be resolved. Food fortification is the most widely used approach to increase the dietary intake of iron. Cereals, legumes, and vegetables are introduced to infant’s nutrition in the period of four and six months to supplement breast milk and infant foods to support rapid growth and development (Swanson, 2003). Infant cereals are commonly fortified with vitamins and minerals in order to achieve dietary reference intake (DRI). However, in fortification not only the quantity but also the stability and bioavailability of minerals are very important (Benito and Miller, 1998). Bioavailability can be defined as the extent to which a nutrient is capable of being absorbed to be utilized within the body. A large number of factors are likely to influence the proportion of a nutrient absorbed from a particular food. These can be related to the form of iron (heme or non-heme), characteristics of the foodstuff (presence of inhibitors such as phytic acid, polyphenolic compounds, or enhancers like ascorbic acid), and gastrointestinal conditions (Benito and Miller, 1998; Hurrell, 1997; Garrick and Garrick, 2009). Phytic acid (myo-inositol-6-phosphate), an antinutritional factor, is found primarily in unrefined cereal grains, legumes, and oil seeds. Under physiological conditions, it is negatively charged, thus strongly chelates with cations such as zinc, calcium, and iron
Nutrition & Food Science Vol. 40 No. 5, 2010 pp. 485-493 # Emerald Group Publishing Limited 0034-6659 DOI 10.1108/00346651011076992
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and causes the formation of insoluble salts of these minerals with poor absorption characteristics and low bioavailability (Abebe et al., 2007). Hence, to ensure absorption of these minerals from a meal, it is important to consider the molar ratios of phytate:mineral of each plant-based food. The reported desirable phytate:mineral molar ratios, for mineral absorption, is less than 1 for phytate:iron and less than 18 for phytate:zinc (Chan et al., 2007). Therefore, phytic acid becomes an important antinutritional factor in cereal-based infant foods by decreasing the bioavailability of iron and also other minerals (Abebe et al., 2007; Chan et al., 2007). In this respect, the objective of this research was to determine iron bioavailability by using in vitro method in commercial infant cereals. These infant cereals are fortified with vitamins and minerals plus iron in order to achieve DRI. Studied samples are famous brands in Turkey and also in Europe. With this aim iron contents, in vitro iron bioavailability, and phytic acid contents of six fortified infant cereals were investigated. Material and methods Chemicals Ferrozine (Sigma P-9762), pepsin (Sigma P-7000), Porcine pancreatin (Sigma P-1750), bile extract (Sigma B-8631), phytic acid dodecasodium salt hydrate (Sigma P-0109), and dialysis bag (Sigma D 9777) were purchased from Sigma-Aldrich. Deionized water was used in all experiments. Materials Six commercial types of infant cereals based on different cereal flours and fortified with several vitamins and minerals plus iron were purchased from various supermarkets in I_zmir, Turkey. Samples were prepared according to declaration by the manufacturer, which was displayed on the product label. Analyzed infant cereals and their information are shown in Table I. Methods All analysis was performed as three replicates and two parallels and averages were calculated. Total iron determination Iron of infant cereals was extracted according to the rapid method used by Kosse et al. (2001) and the iron content of the samples was analyzed according to ferrozine method modified by Kapsokefalou and Miller (1991). In vitro determination of iron bioavailability The relative iron bioavailability of the samples was determined by the in vitro procedure proposed by Miller et al. (1981) and Kapsokefalou and Miller (1991), and modified by Haro-Vicente et al. (2006). After the in vitro digestion the ferrous and ferric iron concentrations of dialysates and retentates were measured using a method modified by Kapsokefalou and Miller (1991). Calculations Calculations were made according to Kapsokefalou et al. (2005) and Haro-Vicente et al. (2006).
Sample no. 1
Type
Major ingredient
Multi-cereals with milk
Partially hydrolyzed cereal flours (oat, rice, wheat, barley, millet, maize, and rye), whey protein, milk powder, vegetable oil, and maltodextrin Hydrolyzed cereal flours (wheat, maize, rice, oat, rye, barley, millet, white millet), skim milk powder, lactose, maltodextrin, vegetable oil Hydrolyzed cereal flours (wheat, maize, rice, oat, rye, barley, millet, and white millet), skim milk powder, molasses, lactose, vegetable oil, casein, and maltodextrin Rice, hydrolyzed rice, and maize semolina Cereal flours: hydrolyzed wheat, wheat, rice, barley, rye, and maize Rice flour, grinded sugar, lactose, skimmed milk powder, serum proteins, vegetable oil, and lactoferrin
2
Eight cereals with milk
3
Eight cereals with molasses
4
Preterm cereals With rye and maize
5
6
Rice flour with milk and sugar
Dialyzable ferrous iron (DFI) ¼
Total dialyzable iron (TDI) ¼
Zinc content (mg/100 g)
Vitamin C content (mg/100 g)
2.1
60
Iron fortificant
Effect of phytic acid in cereals
Iron diphosphate
487 5
25
Iron sulfate
5
25
Iron sulfate
–
44
–
50
Iron diphosphate Iron diphosphate
4.5
25
Not declared
½Feþ2 D ðg=mlÞ dialysate volume ðmlÞ 100 iron in original sample ðgÞ
½Total ironD ðg=mlÞ dialysate volume ðmlÞ 100 iron in original sample ðgÞ
½Feþ2 D ðg=mlÞ dialysate volume ðmlÞ þ½Feþ2 R ðg=mlÞ retentate volume ðmlÞ 100 Ferrous solubleiron ðFSIÞ ¼ iron in original sample ðgÞ ½Total ironD ðg=mlÞ dialysate volume ðmlÞ þ½Total ironR ðg=mlÞ retentate volume ðmlÞ Total soluble iron (TSI) ¼ 100 iron in original sample ðgÞ
Table I. Types of infant cereals and their major ingredients, zinc, vitamin C content, and iron fortificants which are displayed on the label
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where ½Feþ2 D is the ferrous iron concentration in the dialysate, ½Feþ2 R is the ferrous iron concentration in the retentate, and ½Total ironD and ½Total ironR are ferrous plus ferric iron concentrations in dialysate and retentate, respectively. Phytic acid determination Phytic acid contents of infant cereals were determined according to the method adopted by Talamond et al. (1998). Statistical analysis Data on DFI, TDI, FSI, and TSI measurements and phytic acid contents were analyzed by one-way ANOVA (SPSS 13.0). Tukey’s multiple range tests was used to determine significant differences among means ( p < 0.05) and a Pearson’s correlation analysis was carried out for the analysis of interaction between total iron content and dialyzable iron percentage. Results Total iron Total iron content of the samples and declared iron contents on the labels of the samples were given in Figure 1. As can be seen from the figure, sample 3 has the highest iron content (12 1 mg/100 g) whereas sample 6 has the lowest iron content (1.34 1 mg/100 g). Total iron content of samples 1 and 3 were determined 35.1 and 20.0 percent higher than the declared iron amounts on the nutrition facts panel of these samples, respectively. On the other hand the content of total iron in samples 4, 5, and 6 were 54.4, 57.5, and 73.2 percent lower than the declared values on the nutrition facts panel of these samples, respectively. Bioavailability of iron DFI, FSI, TDI, and TSI percentages of the samples were determined for the prediction of iron bioavailability and these results were given in Table II. DFI was not detected for samples 1, 2, and 3. DFI percentages of samples 4, 5, and 6 were 2.51 0.38, 4.12 1.52, and 0.50 0.08, respectively. Sample 5 showed the highest DFI than those of samples 4 and 6 ( p < 0.05).
Figure 1. Analyzed iron contents of infant cereals versus declared iron amounts on the labels
FSI of the samples was determined between 0.23 and 15.98 percent. The percentages of FSI for samples 1, 2, and 3 were lower (p < 0.05) than those of the other samples. Sample 6 presented the highest percentage of FSI (15.98 7.95) (p < 0.05), while the results for samples 4 and 5 (6.35 2.50 and 8.60 2.20 percent, respectively) were similar. According to these results lower than 50 percent of FSI was dialyzable for samples 4 and 5. However, for sample 6, only a small fraction of FSI (approximately 3 percent) was dialyzable. For all samples TDI was in the range of 0.80-9.81 percent. The highest TDI (9.81 2.02) was obtained for sample 6 (p < 0.05) whereas the lowest percentage was for sample 3 (0.80 0.77).
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Phytic acid Table III shows the phytic acid levels and phytate:iron molar ratios in the analyzed infant cereals. Phytic acid concentrations of the samples were determined within the range of 118-161 mg/100 g. The phytic acid concentrations of samples 3, 5, and 6 were significantly higher (p < 0.05) than those of the other samples, whereas sample 2 showed the lowest phytic acid content (118 7 mg/100 g). Phytate:iron molar ratios of all the samples were equal to or higher than 1. The highest ratio was observed for sample 6 (9.89) whereas the lowest ratio was obtained for sample 2 (1.00). Discussion Total iron content As can be seen from Figure 1, the determined iron contents of samples were different (higher or lower) than the declared iron contents on the nutrition facts labels of the Sample no.
DFI (%)
FSI (%)
TSI (%) 0.82a 1.52a 1.45a 1.60a 3.11a 14.74b
Table II.
Notes: Results are given as mean standard deviation; values with different superscripts within a column are significantly different: p < 0.05; ND: not detected
DFI, FSI, TDI, and TSI percentages of infant cereals analyzed
1 2 3 4 5 6
Sample no. 1 2 3 4 5 6
ND ND ND 2.51 0.38b 4.12 1.52c 0.50 0.08a
0.23 0.28 0.36 6.35 8.60 15.98
0.04a 0.36a 0.32a 2.50a,b 2.20b 7.95c
TDI (%)
Phytic acid content (mg/100 g) 146 118 161 126 158 156
19b 7a 11c 8ab 15c 3c
1.19 1.03 0.80 2.78 6.25 9.81
0.43a 0.59a 0.77a 0.19a 1.33b 2.02c
6.28 12.19 11.14 7.57 12.17 111.92
Phytate:iron molar ratios 1.61 1.00 1.13 2.34 3.15 9.89
Notes: Results are given as mean standard deviation; values with different superscripts within a column are significantly different: p < 0.05
Table III. Phytic acid concentrations and phytate:iron molar ratios of infant cereals
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samples. This difference might be due to declared iron amounts on the nutrition facts panel was based on calculation instead of analytical procedure. Another possible reason was incorrect declaration of iron amounts without considering the other ingredients which could also contain iron. Bioavailability of iron All the measurements except DFI generally give information about suitability of the type of iron fortificant used that is more dialyzable and more soluble form in the gastrointestinal system conditions. Different in vitro studies related to evaluation of the bioavailability of iron revealed that the percentage of DFI rather than TDI is well correlated with in vivo studies (Haro-Vicente et al., 2006; Kapsokefalou et al., 2005; Drago and Valencia, 2004; de Souza Nogueira et al., 2005; Argyri et al., 2009). Type of iron used in the fortification process is very important for the point of bioavailability. In general, the higher the solubility of iron compound, the higher the bioavailability of iron in the gastrointestinal system (Benito and Miller, 1998; Hurrell, 1997). In our study, total solubility of iron sulfate and iron diphosphate was not found to be significantly different ( p < 0.05) (Tables I and II). Although samples 1, 4, and 5 contain ferrous diphosphate as a fortificant, DFI is not detected for sample 1. This can be explained by the presence of other ingredients in the formula. All the samples except 4 and 5 contain whey powder and/or lactose as ingredients. The possible interaction between these ingredients and iron can affect dialyzability of ferrous iron. Similarly, de Souza Nogueira et al. (2005) revealed that the presence of casein in the infant formula plays an important role in reducing and inhibiting the dialyzability of iron. Vitamin C is known as the most powerful enhancer of iron bioavailability. Vitamin C contents of the infant cereals, which are declared on their labels, are changed between 25 and 60 mg/100 g. The reason of higher DFI obtained for samples 4 and 5 might be explained by their higher vitamin C contents (44 and 50 mg/100 g, respectively) than those of the other samples except sample 1 which has the highest vitamin C content. The explanation of this exception may be due to possible interactions between iron and the other ingredients that samples 4 and 5 do not contain, such as millet, milk powder, maltodextrin, and vegetable oil. For all the samples, a multi-mineral fortification procedure was applied by the manufacturers. Samples 1, 2, 3, and 6 contain also zinc sulfate as another mineral fortificant. It was reported that multiple fortification of foods with zinc and iron caused zinc to affect iron absorption related to total content of these minerals (Ca´mara et al., 2007; Olivares et al., 2007). In our study, Fe:Zn molar ratios for samples 1, 2, 3, and 6 were 4.29, 2.34, 2.81, and 0.35, respectively and available iron was not determined in samples 1, 2, and 3 that contain zinc. There was no correlation (r ¼ 0.463, p < 0.05) between the total iron content of the infant cereals and the percentage of DFI. This result agreed with the literature that mentioned the quantity of total iron present in food did not influence the content of dialyzable iron (Drago and Valencia, 2004). According to Table I only a small percentage of FSI was dialyzable. This could be due to the type of iron fortificant used, presence of possible ingredients that interact with iron, and/or phytic acid contents of infant cereals. Similar results for DFI and FSI were also reported by different researchers (Haro-Vicente et al., 2006; Kapsokefalou et al., 2005). Haro-Vicente et al. (2006) suggested that the small percentage of DFI could
be owing to binding of ferrous iron to some ligands in food that lead to formation of high molecular weight iron complexes, which cannot transport across the membrane. Phytic acid For phytic acid content it is difficult to compare the results of our study with other studies because apart from the type of cereals used, phytic acid content also depends on the industrial treatments applied such as soaking, fermentation, milling, roasting, or germination and amount of endogenous phytase activity (Liu et al., 2008). In our study, cereal composition of the analyzed infant cereals was also different from each other. Samples 1, 2, 3, and 5 were composed of the mixture of different cereals (oat, rice, wheat, barley, millet, maize, and rye) with different amounts, whereas samples 4 and 6 were based primarily on rice. According to literature not only the phytic acid concentration but also the estimation of phytate:mineral molar ratio is a valuable tool in predicting the inhibitory effect of phytate on the bioavailability of minerals. Phytate:iron molar ratio >1 is an indication of poor iron bioavailability (Chan et al., 2007; Mitchikpe et al., 2008). According to our results, both samples 4 and 5 have the highest iron bioavailability and phytate:iron molar ratios than samples 1, 2, and 3. This might be due to the presence of other factors rather than phytate that cause increasing or decreasing effect on iron bioavailabilities. Tako et al. (2009) and Ariza-Nieto et al. (2007) reported that polyphenols had greater inhibitory effects on iron bioavailability as in comparison to phytate. Furthermore, in two different studies it was reported that in the presence of the relatively high concentration of ascorbic acid in the test meal showed no significant effect of phytic acid on iron bioavailability (Davidsson, 2003; Lestienne et al., 2005). Our results are also in good agreement with this result. This might be also due to the presence of higher percentage of vitamin C in samples 4 and 5 than those of the other samples and the presence of zinc with different amounts in samples 1, 2, 3, and 6. Conclusions The data presented here showed that in most of the analyzed infant cereals the bioavailability of iron was under detectable limits and/or very low. The study led us to conclude that the phytate:iron molar ratio is not the major inhibitory factor on iron bioavailability for the analyzed infant foods. Other possible factors might be the type of iron fortificant added, synergistic effect of Vitamin C and/or inhibitory effect of Zn, and possible interactions of iron with other ingredients in the formula such as phenolic compounds, lactose, and whey proteins. The applied in vitro method cannot be used alone for an important decision taken by industry for new product development that also in vivo studies are required. However, it can give an idea for increasing the bioavailability of iron, by effective control of the composition of cereals and preparation of optimum formulation ratios. References Abebe, Y., Bogale, A., Hambidge, K.M., Stoecker, B.J., Bailey, K. and 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, Vol. 20, pp. 161-8. Argyri, K., Birba, A., Miller, D.D., Komaitis, M. and Kapsokefalou, M. (2009), ‘‘Predicting relative concentrations of bioavailable iron in foods using in vitro digestion: new developments’’, Food Chemistry, Vol. 113, pp. 602-7.
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