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Am J Clin Nutr 89: 539-544, 2009. First published December 23, 2008; doi:10.3945/ajcn.2008.27026
 American Journal of Clinical Nutrition, doi:10.3945/ajcn.2008.27026
Vol. 89, No. 2, 539-544, February 2009
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© 2009 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

Optimization of a phytase-containing micronutrient powder with low amounts of highly bioavailable iron for in-home fortification of complementary foods

Barbara Troesch1,2,3, Ines Egli1,2,3, Christophe Zeder1,2,3, Richard F Hurrell1,2,3, Saskia de Pee1,2,3 and Michael B Zimmermann1,2,3

1 From the Laboratory for Human Nutrition, Institute of Food Science and Nutrition, ETH, Zurich, Switzerland (BT, IE, CZ, RFH, and MBZ); the World Food Program, Rome, Italy (SdP); the Friedman School of Nutrition Science and Policy, Tufts University, Boston, MA (SdP); and the Division of Human Nutrition, Wageningen University, Wageningen, Netherlands (MBZ).

2 Supported by the Foundation Nutrition Industry (Basel, Switzerland), established by DSM Nutritional Products Ltd, and the ETH (Zürich, Switzerland).

3 Reprints not available. Address correspondence to B Troesch, Laboratory for Human Nutrition, ETHZ, Zürich ETH Zentrum, Schmelzbergstrasse 7, LFV D19.3, 8092 Zürich, Switzerland. E-mail: barbara.troesch{at}ilw.agrl.ethz.ch.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: In-home fortification of complementary foods with micronutrient powders containing low amounts of iron may be potentially safer than powders containing high amounts of iron. However, low iron doses have little nutritional effect, unless iron absorption is high.

Objective: The objective was to maximize iron absorption from a low-iron micronutrient powder for in-home fortification by testing combinations of iron as NaFeEDTA, ascorbic acid, and a microbial phytase active at gut pH. In addition, a recently proposed enhancer of iron absorption, L-{alpha}-glycerophosphocholine (GPC), was tested.

Design: In 6 separate iron-absorption studies using a crossover design, women (n = 101) consumed whole-maize porridge fortified with 3 mg stable isotope–labeled FeSO4 or NaFeEDTA with different combinations of enhancers added to the meals at the time of consumption. Incorporation of iron isotopes into erythrocytes 14 d later was measured.

Results: The addition of phytase when iron was present as either NaFeEDTA or FeSO4, with or without ascorbic acid, significantly increased iron absorption. The combined addition of phytase, ascorbic acid, and NaFeEDTA resulted in an absorption of 7.4%, compared with an absorption of 1.5% from FeSO4 without enhancers in the same meal (P < 0.001). The addition of ascorbic acid did not significantly increase iron absorption from NaFeEDTA, and the addition of calcium did not significantly inhibit iron absorption from NaFeEDTA in the presence of ascorbic acid. The addition of L-{alpha}-glycerophosphocholine did not significantly increase iron absorption.

Conclusion: Optimization of the micronutrient powder increased iron absorption from a highly inhibitory meal {approx}5-fold. This approach may allow for effective, untargeted in-home fortification of complementary foods with low amounts of highly bioavailable iron.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Iron deficiency anemia is a major public health problem, and infants are particularly vulnerable (1). Provision of sufficient dietary iron to this age group is a challenge, and in-home iron fortification of complementary foods is a promising approach (2). Micronutrient powders, such as Sprinkles (Sprinkles Global Health Initiative, Toronto, Canada), MixMe (DSM Nutritional Products Ltd, Basel, Switzerland), or MoniMix (Renata Ltd, Dhaka, Bangladesh), which are added to complementary food after cooking, can be an effective approach for providing additional dietary iron (2). The home-fortification approach using a micronutrient powder containing 12.5 mg Fe as ferrous fumarate given daily reduces the prevalence of iron deficiency anemia in infants (3). However, untargeted iron supplementation of young children in areas of high malaria endemia may increase morbidity and mortality (4–6).

In-home fortification of complementary foods with micronutrient powders containing low amounts of iron may be potentially safer than using powders containing high amounts of iron (7). However, low iron doses will have little nutritional effect unless iron absorption is high. Complementary foods in developing countries are typically based on starchy staples often combined with legumes (8). The high amounts of phytic acid (myo-inositol-6-phosphate) in these diets strongly reduce the absorption of iron. Phytate can be enzymatically degraded before consumption (8–12). One previous study has suggested that a phytase that is activated at gut pH may be effective in increasing iron absorption when added to a meal just before consumption (13). A chelated form of iron, NaFeEDTA, can provide well-absorbed iron even in the face of high-phytate diets (14). Recent guidelines suggest that EDTA is safe at intakes of up to 1.9 mg · kg body wt–1 · d–1 (15). Ascorbic acid is also a potent enhancer of iron absorption from high-phytate foods (16). Whether the combination of ascorbic acid and EDTA would have additional benefits on iron absorption from a high-phytate meal is unknown; in a noninhibitory meal, there appears to be no benefit of using EDTA together with ascorbic acid (17). Recently, L-{alpha}-glycerophosphocholine (GPC) has been proposed to enhance iron absorption (17). The aim of the study was to test different combinations of enhancers in an attempt to maximize iron bioavailability from a low-iron micronutrient powder in a high-phytate meal.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Apparently healthy young women (n = 101) were recruited from the student and staff populations at the ETH Zurich and the University of Zurich between January and April 2008. The sample size of 16 women per group is sufficient to detect an intrasubject difference of 30% in iron absorption with an {alpha} level of 0.05 and a β level of 0.20. Exclusion criteria were pregnancy and lactation as well as known gastrointestinal or metabolic disorders. No medication (except oral contraceptives) or vitamin and mineral supplements were allowed during the study. Subjects who regularly consumed vitamin and mineral supplements were asked to discontinue supplementation 2 wk before the start of the study. None of the subjects had donated blood <4 mo before the start of the study. Written informed consent was obtained from all subjects. The study protocol was approved by the ethics committee at the ETH (Zurich, Switzerland).

Study design
The study consisted of 6 separate iron-absorption studies, and the participants were randomly assigned to 1 of 6 groups. They were fed a maize porridge fortified with a micronutrient powder (Table 1) containing either ferrous sulfate or NaFeEDTA with different combinations of inhibitors and enhancers (Table 2). They each consumed 2 meals in a crossover design (days 1 and 2). The isotopically labeled compounds were added directly to the test meals at the time of feeding.


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  		TABLE 1. Composition of the core powder1

 

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  		TABLE 2. Fractional iron absorption from the different test meals1

 
On day 1, the participants’ heights and weights were measured, and their eligibility for the study was assessed with a short questionnaire. All test meals were served between 0700 and 0900 in the morning after an overnight fast under standardized conditions and with close supervision. Other than 300 mL high-purity water served with the meals, no food or drink was allowed for 3 h after consumption of the meals. Fasting venous blood was drawn into EDTA-treated tubes at baseline and on day 16. Calculation of iron absorption was based on the shift in the isotopic ratios after a 14-d incorporation period as described in detail by Walczyk et al (18).

Preparation of stable-isotope labels
Isotopically labeled 57FeSO4 and 58FeSO4 were prepared from isotopically enriched elemental iron (Lohmann GmbH, Emmerthal, Germany). Isotopically labeled Na57FeEDTA and Na58FeEDTA were prepared by using the method described by Loots et al (19). Isotopic enrichment was 97.8% for Na57FeEDTA, 91.9% for Na58FeEDTA, and 95.4% and 93.0% for 57FeSO4 and 58FeSO4, respectively.

Test meals
The basis for the test meals for all groups consisted of porridge made from 60 g whole maize and 200 g high-purity water (18 M{Omega}) sweetened with 5 g sugar. The amount of maize provided in the meal was based on the amount typically served to preschool children in South African school-lunch programs. The porridge was cooked the day before the study. Once it had cooled to room temperature, evaporated water was replaced and 265-g samples were weighted into porcelain bowls and stored at 4°C overnight and then heated to 34–38°C just before consumption. Core powder mix (500 mg; DSM Nutritional Products Ltd, Basel, Switzerland) (Table 1), 3 mg labeled iron (57Fe or 58Fe), and the inhibitors and/or enhancers to be studied (Table 2) were added directly to the warm test meal. Ascorbic acid, tricalcium phosphate, and phytase were obtained from DSM Nutritional Products Ltd. GPC (Lipoid GPC X; Lipoid GmbH, Ludwigshafen, Germany) contained 90% active ingredient. In a previous study (17), GPC was added to a moderately inhibitory meal at a molar ratio of GPC to iron of 2 to 1 based on the assumption that its enhancing capacity was similar to that of ascorbic acid. Because the meal we used in this study was much more inhibitory than the one used previously, we chose to add GPC at a ratio of 4 to 1.

With each meal, the participants received 300 mL high-purity water. When the meal was served to the participants, they were instructed to eat the whole meal, to rinse the bowl with some water, and to drink all of the water. The time needed to finish the test meal and water was recorded.

Measurements of iron status and isotope composition in blood
Hemoglobin was measured in whole blood on the day of collection by using an automated Coulter counter (AcT8 Counter; Beckman Coulter, Krefeld, Germany) with 3-level control materials provided by the manufacturer. Serum ferritin was measured with an IMMULITE automatic system (DPC Bühlmann GmbH, Aschwil, Germany).

Anemia was defined as a hemoglobin concentration <120 g/L, and iron deficiency was defined as a serum ferritin concentration <15 µg/L (20). Each isotopically enriched blood sample was analyzed in duplicate for its iron isotopic composition under chemical blank monitoring. Whole-blood samples were mineralized by using a mixture of HNO3 and H2O2 and microwave digestion, which was followed by separation of the sample iron from the matrix by anion-exchange chromatography and a solvent-solvent extraction step into diethyl ether (18). All isotopic analyses were performed by negative thermal ionization–mass spectrometry (MAT 262; Finnigan MAT, Bremen, Germany).

Calculation of iron absorption
Circulating iron was calculated on the basis of the blood volume, which was estimated from height and weight according to Brown et al (21). For calculation of fractional absorption, 80% incorporation of the absorbed iron into red blood cells was assumed (18).

Characteristics of phytase
The phytase used was an enzyme derived from a genetically modified culture of Aspergillus niger (DSM FS Phytase 20.000 G; DSM, Delft, Netherlands). The phytase has 2 pH optima, one at pH 5 with an activity defined as 100% and the other at pH just <3 with an activity level of {approx}60% (DSM Phytase 20.000 G, application data sheet; DSM Nutritional Products, Basel, Switzerland). The quantity of phytase added to the meal was calculated assuming the following: 1) a highly inhibitory meal may contain up to 1 g phytate, and 2) a residence time in the stomach of {approx}60 min. Phytase activity is measured as the amount of enzyme that liberates 1 µmol inorganic phosphorus/min and is called a phytase unit (FTU). Because the phytase activity at gastric pH should be 50–60%, 8 FTU was necessary to adequately degrade 1 µmol phytate ({approx} 0.7 mg phytic acid). Therefore, we estimated that 190 FTU was needed per serving to achieve the necessary degradation of 1 g phytic acid.

Simulation of phytase activity in the meal and the stomach
To estimate phytate degradation in the test meal, the phytate content of the maize porridge was measured in set intervals over 1 h after phytase was added at a pH of 7, 3, 2, and 1. The amounts of maize porridge and added phytase were the same as those used in the absorption studies. The phytate content was measured by using a modified method of Makower (22), and inorganic phosphate was determined according to Van Veldhoven and Mannaerts (23).

Statistical analysis
The statistical analyses were conducted by using Microsoft Office Excel 2003 and SPSS software (version 16.0; SPSS Inc, Chicago, IL). Data not normally distributed were log transformed for analysis. Iron absorption was presented as geometric means and compared by using paired and nonpaired t tests. To compare absorption between groups, the results were corrected to a value corresponding to a serum ferritin concentration of 40 µg/L (24). Unpaired t tests were also used to check for differences in iron status and body mass index (BMI) between the 6 study groups. BMI was calculated as weight (in kg) divided by height squared (in m). Regressions were used to test for associations between the time needed to finish the meals containing phytase and iron absorption from those meals. P values < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Participants
The mean (±SE) hemoglobin concentration of the participants was 128 ± 0.1 g/L; the geometric mean (±SE) serum ferritin concentration was 27.9 ± 3.6 µg/L. Hemoglobin and serum ferritin concentrations did not differ significantly between the 6 study groups. One of the subjects had iron deficiency anemia, and 21 of the subjects were iron deficient but not anemic. The mean (±SD) BMI of the subjects was 20.3 ± 1.6, and no significant differences in BMI were observed between the 6 groups (P > 0.05).

Composition of the test meals
The mean iron content of the unfortified maize flour was 1.25 mg/100 g, which provided {approx}0.8 mg native iron per portion of maize porridge. The mean phytate content was 0.60 g/100 g maize flour, or {approx}0.36 g for each test meal. Considering both the native iron content of the meal and the content of fortification iron (3 mg) in the powder, the molar ratio of phytate to iron in the test meals was {approx}8:1—a highly inhibitory ratio (11).

Iron absorption from the different test meals
The fractional iron absorptions from the different test meals are summarized in Table 2. The main findings were as follows:

1) The addition of phytase when iron was present as either NaFeEDTA or FeSO4 significantly increased iron absorption.
2) The addition of phytase when iron was present as NaFeEDTA and the meal contained ascorbic acid also significantly increased iron absorption.
3) The addition of calcium did not significantly inhibit iron absorption from NaFeEDTA in the presence of ascorbic acid; the molar ratio of ascorbic acid to iron to calcium in the test meal was 1:0.2:15.
4) The addition of ascorbic acid did not significantly increase iron absorption from NaFeEDTA; the molar ratio of ascorbic acid to iron in the test meal was 5:1.
5) The addition of GPC did not increase iron absorption from FeSO4 in the test meal when given at a molar ration of GPC to iron of 4:1.

There was a significant difference in iron absorption between the test meals without added enhancers (study 6, meal B; Table 2) and with the 3 enhancers (study 4, meal A; Table 2) corrected to a serum ferritin concentration of 40 µg/L (P < 0.001) (Figure 1). The meals with FeSO4 that did not contain phytase were significantly less well absorbed than were the meals containing iron as FeSO4 in the presence of phytase or when iron was added as NaFeEDTA (P < 0.05). Iron absorption from the meals containing FeSO4 with phytase was not significantly different those where the iron was given as NaFeEDTA. There were no significant differences in iron absorption between the different meals containing iron as NaFeEDTA.


Figure 1
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  		FIGURE 1. Comparison of fractional iron absorption between the meals from the different studies. The black lines show the mean absorption for each meal. Different levels of absorption from maize porridge were logarithmically transformed. The results of the statistical comparisons are in the text. GPC, L-{alpha}-glycerophosphocholine.

 
In vitro simulation of phytase activity in the meal and the stomach
Before the addition of phytase, the meal contained 0.125 g phytic acid/100 g. Measurements were taken 10, 20, 30, and 60 min after phytase was added to the test meal: 1) at pH 7, the phytic acid concentrations were 0.078, 0.044, 0.024, and 0.012 g/100 g; 2) at pH 3, the values were 0.108, 0.102, 0.097, and 0.072 g/100 g; and 3) at pH 2, the values were 0.106, 0.097, 0.085, and 0.066 g/100 g. Phytase showed no discernable activity at pH 1.

Influence of time on iron absorption from the test meals with phytase
On average, it took the participants 30 min to finish their meal. Regressions showed no correlations between the time needed to finish the meal containing phytase and iron absorption from the meal, even after adjustment for absorption from the paired meal without phytase and the participant's serum ferritin concentration.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In study 4, the addition of microbial phytase and ascorbic acid increased the absorption of 3 mg Fe as NaFeEDTA from a highly inhibitory meal to 7.4%. This is a more than 5-fold increase in iron absorption compared with the 1.5% absorption of iron as FeSO4 without enhancers from the same meal in study 6 (P < 0.001). Because the combination of phytase, ascorbic acid, and EDTA should also increase absorption of the native iron in the maize to a similar level, the total amount of absorbed iron from the fortified meal would be {approx}0.28 mg. Assuming a similar increase in absorption, the use of the micronutrient powder containing the 3 enhancers, even when added to highly inhibitory complementary foods, could significantly contribute to ensuring that iron needs are met in infants and young children.

In a previous human study, Sandberg et al (13) found that the addition to a test meal just before consumption of a phytase active at neutral and gastric pH increased iron absorption of 3.7 mg Fe as FeSO4 from 14.3% to 26.1%. This increase corresponds to an absorption ratio of 1.98, which is similar to the ratio of 1.71 found in our study using FeSO4 (study 5). The meal used by Sandberg et al (13) contained smaller amounts of phytate and a higher amount of added phytase per meal than did the meal in our study. The increase in iron absorption with the addition of phytase in both Sandberg et al's study and our study was substantially less than that achieved in other studies in which the amount of phytate was reduced to negligible levels by other means and iron absorption increased 4–12-fold (11, 25). This suggests that the phytic acid in the meal was not completely degraded by the phytase.

There could be several reasons for this. It is possible that phytase was rapidly inactivated in the stomach, if the gastric pH approached 1. However, this was unlikely because a standard breakfast quickly raises the gastric pH above 2, and maintains the pH between 2 and 3 for {approx}1 h, and phytase is partially active at pH 2–3 (26). Another explanation could be the presence of calcium in the powder. Calcium can impair phytate degradation by phytases (27–29). Hallberg et al (30) suggested that calcium forms complexes with phytic acid that protect it from enzymatic degradation, but do not prevent it from binding iron and inhibiting its absorption. Another potential explanation is that meal residence time in the stomach and proximal duodenum is insufficient to allow the phytase to act long enough to degrade the phytate in a maize porridge below the critical phytate to iron ratio of >0.4:1 (11, 25).

The phytase tested here offers clear advantages to adding phytase to degrade phytate in foods during processing before consumption. In developing countries, most infants and children are not fed commercially produced complementary foods (31). Therefore, dephytinization would have to take place during meal preparation at the household level. Depending on the exact type of phytase used as well as the temperature and the pH of the meal, the time needed to degrade phytate varies; however, the food must typically be left at room temperature or above for >1 h, which increases the possibility of food-borne diseases (32–34).

EDTA is an iron chelator that enhances iron absorption from inhibitory meals by reducing iron binding to phytic acid at a low pH. The addition of EDTA increases iron absorption from meals of varying composition 2–4-fold (35–41). With our test meal, the total amount of iron (native and fortified) was {approx}3.8 mg at a molar ratio of EDTA to iron of {approx}0.8:1. This molar ratio of EDTA to iron is in the range that has been shown to maximally increase iron absorption from high-phytate meals (40). Despite this, the addition of phytase to the test meal containing iron as NaFeEDTA significantly increased iron absorption (by 23%). Thus, although EDTA enhances iron absorption from foods high in phytic acid, it does not provide complete protection from the inhibitory effects of phytic acid (35).

A molar ratio of ascorbic acid to iron of 4:1 can maximally enhance the absorption of nonchelated iron compounds from inhibitory meals (42–44). In our study, the addition of 60 mg ascorbic acid in the presence of iron as NaFeEDTA (a molar ratio of ascorbic acid to iron of 5:1) did not significantly increase iron absorption from the maize porridge. These data support the findings of MacPhail et al (38), who reported that the addition of 50 mg ascorbic acid to a maize porridge did not increase iron absorption in the presence of EDTA, but the addition of 100 mg ascorbic acid led to a significant 2-fold increase. Therefore, it is possible that a higher molar ratio of ascorbic acid to iron might have enhanced iron absorption in the presence of EDTA in our maize-based meal. Although ascorbic acid showed no additional enhancing effect in the presence of EDTA in the present study, its presence in the powder may still prove advantageous if the powder is added to other foods. For example, reducing the phytate content alone is not sufficient to enhance iron absorption when complementary foods are reconstituted with milk or contain milk (45). In this setting, the addition of ascorbic acid is beneficial. Polyphenols reduce iron absorption from EDTA (38), but the addition of ascorbic acid can at least partially overcome their inhibiting effect (46).

As expected, the 200 mg Ca added as tricalcium phosphate did not significantly decrease iron absorption from NaFeEDTA. Although findings on the effect of calcium on iron absorption are equivocal (30), recent stable-isotope studies by our group also showed no inhibition of iron absorption when 200 mg Ca was added to an instant drink with an ascorbic acid to iron ratio of 2:1 and 4:1 in the absence of EDTA (R Hurrell, personal communication, 2008). GPC is commercially available and occurs naturally in a wide range of foods (47). It was recently found to enhance nonheme-iron absorption when added at a molar ratio of 2:1 to iron in a moderately inhibitory meal (17). In the present study, we found that GPC did not increase iron absorption when added at a 4:1 ratio with iron to a highly inhibitory meal. It is possible that differences in the food matrix between the 2 studies explain the discrepant results.

Our findings suggest that the optimized phytase-containing micronutrient powder studied may be a promising approach to fortifying even highly inhibitory complementary foods with low amounts of iron. However, several questions remain. Legal issues will have to be resolved, because in many countries the use of phytase from a genetically modified Aspergillus niger could be restricted by national legislation. The French food safety agency has approved its use for bread making, and the US Food and Drug Administration has classified it as Generally Recognized as Safe–self-affirmed. The manufacturer of the phytase used in the present study cannot currently provide a reliable cost estimate for human use, because the final cost will be based on production demand and purity standards. Future studies should test potential ways to increase the phytase's efficacy at degrading phytic acid in vivo, such as by prolonging stomach transit time, and to determine whether phytase can increase iron absorption from other food matrices.


   ACKNOWLEDGMENTS
 
We thank the subjects for their participation in the study, Marco Eckert for his work on the in vitro simulation of phytase activity, our colleagues for their help with the logistical aspects of the studies, K Kramer (DSM, Basel, Switzerland) and G Steiger (DSM, Vienna, Austria) for their constructive inputs into the study design, DSM for providing the micronutrient powder and ingredients free of charge, and P Jooste (Medical Research Council, Cape Town, South Africa) for providing the maize flour for the study.

The authors’ responsibilities were as follows—BT: wrote the first draft of the manuscript; and BT, IE, CZ, RFH, and MBZ: conducted the data and statistical analyses. All authors helped design the study, execute study, and edit the manuscript. None of the authors declared a conflict of interest.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication September 26, 2008. Accepted for publication November 13, 2008.




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