A complementary food supplement from local food ingredients to enhance iron intake among children aged 6–59 months in Benin

Abstract Nutritious complementary feeding is often not affordable in Benin, and iron deficiency exists. This research aimed at formulating an affordable and sensory acceptable complementary food supplement using local food ingredients to increase iron intake among children aged 6–59 months in Benin. The complementary food supplement was formulated to ensure that 10 g would cover 25% of the estimated average requirements for iron for children aged 6 to 12 months. Adansonia digitata fruit pulp, Moringa oleifera leaf powder, and Cochlospermum tinctorium root powder were used to compose the complementary food supplement, which was mixed with maize and sorghum ogi porridges before being presented to the mothers and children for the acceptability test. The mineral contents of Adansonia digitata fruit pulp in mg/100 g dw were 9.9 ± 0.1 for iron and 0.9 ± 0.1 for zinc. The iron and zinc contents of Moringa oleifera leaf powder and Cochlospermum tinctorium root powder in mg/100 g dw were 34.1 ± 2.2 and 26.8 ± 2.7 and 9 ± 0.0 and 0.9 ± 0.0, respectively. The complementary food supplement contained, in mg/100 g dw, 17.4 ± 1.1 of iron and 1.2 ± 0.1 of zinc. The maize and sorghum ogi porridges enriched with the complementary food supplement at substitution rates of 15% and 16% (in dry weight), respectively, were acceptable to 85% of children for sorghum ogi porridge and 87% for maize ogi porridge. The present study demonstrated the potential of local food ingredients in the formulation of an iron‐rich and acceptable complementary food supplement for children aged 6–59 months in Benin.


| INTRODUC TI ON
Malnutrition, especially micronutrient deficiencies (MNDs), among infants is an important public health problem. Worldwide, MNDs affect an estimated 2 billion people, in particular women and children under 5 years (Bailey et al., 2015;Kassebaum et al., 2014;Saini et al., 2014), which can be related to the low nutrient density of the complementary foods, inadequate complementary feeding practices, child care, and sanitation (Akombi et al., 2017;Issaka et al., 2015). Most complementary foods consumed by infants in many parts of the world are reported to be deficient in essential micronutrients (Adeoti & Osundahunsi, 2017). These complementary foods are usually based on products derived from cereals (Arise et al., 2014;Muhimbula et al., 2011;Van der Merwe et al., 2019) and sometimes fortified with nutrient-dense plant species (Abioye & Aka, 2015;Adejuyitan et al., 2012). Sorghum and maize ogi porridges were identified as the two most popular porridges for food fortification using Moringa oleifera leaf powder and/or Adansonia digitata fruit pulp in Benin (Affonfere, 2018), where MNDs are still affecting many children aged 6-59 months. Among these MNDs, iron deficiency (ID) is the commonest, which remains a major public health burden among children (Akpovwa et al., 2020;Kassebaum et al., 2014;Muriuki et al., 2020). More specifically, in Benin, 72% of under 5-year-old children have anemia (EDS, 2018), possibly due to insufficient iron intake or sufficient intake combined with impaired absorption due to inflammation and infections such as malaria (Baye et al., 2014;Muriuki et al., 2020). Iron deficiency anemia (IDA) is known to be the most common form of anemia and is estimated to be the cause of up to 50% of anemia cases (WHO, 2008). To overcome this issue, many strategies such as infant and child care feeding practices, control of parasitic infections, food fortification, food diversification, nutritional supplementation, and the use of complementary food supplements were proposed (Adetola et al., 2019;Cardoso et al., 2019;Chadare et al., 2019;Das et al., 2013;Nestel et al., 2003;Tripp et al., 2011).
Complementary food supplements include crushable micronutrient tablets and micronutrient sprinkles that are added to food just before feeding (Nestel et al., 2003). Such complementary food supplements do not require any preparation or addition of water before ingestion and can be stored for long periods without refrigeration, allow individual packaging, and can therefore be used effectively in situations with nonoptimal hygiene conditions (Dibari et al., 2012). Moreover, they can be a good solution to deliver micronutrients to children whose parents cannot afford commercial fortified foods. In that framework, the use of low-cost, locally available, and nutritious ingredients to formulate complementary food supplements has been recommended (Kunyanga et al., 2012). Chadare et al. (2017) identified and characterized local food ingredients available in the eight agro-ecological zones of Benin for feeding children aged 6-23 months. Among these food ingredients, Cochlospermum tinctorium root powder, Adansonia digitata fruit pulp, and Moringa oleifera leaf powder are key in terms of their nutritional potential. Specifically, Cochlospermum tinctorium root powder was found to contain up to 70.1 mg/100 g dw of iron (Chadare et al., 2017). Its sauce is known to be consumed by under fiveyear-old children in the Sudanian zone of Benin (Affonfere, 2018).
Nevertheless, its acceptability is limited when mixed with foods, such as porridges, due to its green color (Karim et al., 2013;Salem et al., 2013). Therefore, it is recommended to combine Moringa oleifera leaf powder with other food ingredients to suppress its herbal smell and intense green color (Oyeyinka & Oyeyinka, 2016).
Consequently, the present study aimed at formulating an affordable and sensory acceptable complementary food supplement using Moringa oleifera leaf powder and other local food ingredients to increase iron intake among children aged 6-59 months in Benin.

| Complementary food supplement ingredients
The local food ingredients to formulate the complementary food supplement were Moringa oleifera leaf powder, Cochlospermum tinctorium root powder, and Adansonia digitata fruit pulp. These were chosen for their availability, nutritional composition, and use as food ingredients (Affonfere, 2018;Chadare et al., ,,2009Chadare et al., ,, , 2017Houndji et al., 2013;Kayalto et al., 2013). The average costs of these ingredients are 100 FCFA/100 g for Adansonia digitata fruit pulp (data from Angel's Floor society in Benin), 40 Fcfa/100 g for Cochlospermum tinctorium root powder (data from the local population of Tanguiéta, Benin), and 600 FCFA/100 g for Moringa oleifera leaf powder (data from the Hunger Project, Benin). Moringa oleifera leaf powder and Adansonia digitata fruit pulp were purchased, respectively, at the Hunger Project and Angel's Floor society, two reference centers that produce these ingredients in Benin. Cochlospermum tinctorium root powder was collected among the local population of Tanguiéta (Northern Benin), where the species naturally occurs (Akoègninou et al., 2006). The selected food ingredients and the complementary food supplement were analyzed (in duplicate) for their dry matter, ash, iron (Fe), calcium (Ca), zinc (Zn), magnesium (Mg), phosphorus (P), copper (Cu), sodium (N), manganese (Mn), vitamin C, and total phenolic compounds. Pro-vitamin A, phytate, and tannin contents of the selected food ingredients were collected from the literature.

| Dry matter and ash determination
Dry matter content was assessed by the thermo-gravimetric method according to AOAC (1993). Samples (5 g) for determining dry matter (Equation 1) were weighted in a crucible and dried in an oven at 105°C for 72 hr. Ash content was determined by dry ash in a furnace at 550°C for 8 h (Equation 2).
where P1: crucible weight, P2: weight (sample +crucible) after drying in the oven, and Pe: weight of sample (5 g) where Pi: porcelain weight, Pf: weight (sample +porcelain) after drying in the furnace, and Pe: weight of the sample (5 g).

| Mineral determination
Fe, Ca, Zn, P, Cu, Mg, Na, and Mn contents were determined using atomic absorption spectrometry according to the method described by Pinta (1975). A 5 g sample was placed in a previously weighed porcelain crucible and placed in a muffle furnace at 550ºC for 8 hr.
Ash was collected and dissolved in 5 ml of 20% concentrated chloric acid. The content was filtered through Whatman's ashless filter paper, and the volume was brought to 50 ml with bidistilled water.
From the solution, Fe, Ca, Zn, P, Cu, Mg, Na, and Mn contents were determined using a flame atomic absorption spectrophotometer (Analyst 200 Perkin Elmer). A standard stock solution of each mineral was prepared in parallel by appropriate dilution.

| Vitamin C determination
The vitamin C contents of the selected food ingredients were determined using the 2,6-dichlorophenolindophenol titrimetric method according to International Standard ISO 6557/2 (1984).
The samples were thoroughly mixed with the distilled water, and their filtrates were collected. The filtrate solution (5 ml) was added to oxalic acid (2% v/v) and titrated with freshly prepared 2,6-dichlorophenolindophenol until obtaining a salmon-pink coloration that persisted for at least 5 s.

| Total phenolic compound determination
Total phenolic compounds were determined according to Singleton and Rossi (1965) as described by Kayodé et al. (2007). Total phenolic compounds were extracted from 50 mg of sample in 1.5 ml of HCl/methanol (1% v/v) for 1 hr under continuous stirring at room temperature. The mixture was centrifuged at 5,000 g for 10 min, after which the supernatant was removed. The pellet was re-extracted, and the supernatants were pooled. The extracts (300 µl) were mixed with 4.2 ml of distilled water, 0.75 ml of Folin-Ciocalteu's reagent (Merck, Germany), and 0.75 ml of sodium carbonate solution (20% w/v). After incubation for 30 min, the optical density was measured at 760 nm using a spectrophotometer (JENWAY 6850 UV Vis). Blanks were always freshly prepared, in which Folin-Ciocalteu's reagent was replaced by water to correct for interfering compounds. Gallic acid (New Jersey, USA) was used as standard, and the results were expressed as gallic acid equivalent per 100 g dry weight of samples.

| Formulation of the complementary food supplement
The complementary food supplement formulation was done by the linear programming functionality of Excel Microsoft (Solver-Simplex PL) according to Chakeredza et al. (2008). Formulation objectives were set to minimize the cost of the complementary food supplement and to assure that ten grams would cover 25% of the iron daily estimated average requirements for children aged 6 to 12 months, the age-group with the highest daily estimated average requirements for iron (Ross et al., 2011). The target of a contribution of 25% was chosen as children also consume other sources of iron and to guarantee the acceptability of the complementary food supplement. Next, a constraint was set up for the amount of Moringa oleifera leaf powder, based on its acceptability rate in porridges (Abioye & Aka, 2015) and the daily amount of maize porridge consumed by under 5-year-old children (Affonfere, 2018). A lower limit of 15% was used for each food ingredient for its contribution to the formula. (1)

| Determination of minerals to phytate molar ratios and vitamin C to iron molar ratio
The inhibitory effect of dietary phytate on the bioavailability of minerals from the diet can be estimated through phytate minerals molar ratios (Al Hasan et al., 2016). In the present study, the phytate contents of selected ingredients were collected from the literature and the phytate content of the complementary food supplement was theoretically calculated accordingly. The moles of phytate and minerals were determined by dividing the weights (contents) of phytate and minerals with their atomic weights (phytate: 660 g/mol; Fe: 56 g/ mol; Zn: 65 g/mol; Ca: 40 g/mol). The molar ratios between phytate and minerals were obtained after dividing the mole of phytate with the mole of minerals (Norhaizan & Nor Faizadatul Ain, 2009). The same approach was used for the vitamin C: iron molar ratio. The atomic weight used for vitamin C was 176.12 g/mol.

| Acceptability of the complementary food supplement
The acceptability of the complementary food supplement was assessed by adding the supplement to porridges made from sorghum and maize ogi (Affonfere, 2018). The fortification rates for the acceptability test were defined based on the quantities of sorghum and maize porridges daily consumed by under five-year-old children according to the aforementioned author and the 10 g of complemen- tary food supplement to be consumed daily by the children. Thus, 16% and 15% fortification rates (dry basis) were tested for sorghum and maize ogi porridges, respectively, using five (5) (Mouquet & Treche, 2006). Fifty grams of each fortified porridge was served to the panelist in a white cup, and clean water was provided to rinse the mouth between evaluations. During the consumption of the fortified porridges, the facial expressions of the children were recorded by the investigators using facial hedonic scales of five (05) levels (0: very bad, 1: bad, 2: maybe good or bad, 3: good, and 4: very good) as described by Guinard (2001). These nonverbal cues have previously been recommended (Guinard, 2001) and used in similar studies (Mithamo et al., 2020;Ngoma et al., 2018). The use of nonverbal cues is the practice of choice in the baby food industry (Kevin, 1995). Mothers evaluated the fortified porridges using the sensory attributes under evaluation after their training on how to use a hedonic scale of five (05) levels. The attributes were acidity, color, smell, consistency, and overall acceptability.

| The physicochemical characteristics of enriched and unenriched porridges
The physicochemical characteristics of enriched and unenriched maize and sorghum ogi porridges were determined in triplicate.

| Statistical analyses
Results were expressed as mean ± standard deviation per 100 g sample (dry weight). One-way analysis of variance (ANOVA) with Minitab version 18 was performed for physical characteristics to check the (3) Cover rate ( % ) = Minerals or vitamin content in 10 g dry base CFS * 100 effect of the complementary food supplement on the maize and sorghum ogi porridges. A significant difference was accepted at p < .05.
In case of a difference, the Tukey test was performed to separate means, with a confidence level for significant differences at p < .05.
A significant difference between each sensory attribute scale was checked using the multiple proportion chi-square test. A correlation test (Spearman) was also performed to assess relations between sensory attributes and the overall liking of the enriched porridges.

| Minerals
Moringa oleifera leaf powder was found to be rich in minerals, followed by Cochlospermum tinctorium root powder and Adansonia digitata fruit pulp ( Table 2). The mineral content of Adansonia digitata fruit pulp, expressed as mg/100 g dw, was 9.9 ± 0.1 for iron, 0.9 ± 0.1 for zinc, and 402.2 ± 3.4 for calcium. The mineral content of Moringa oleifera leaf powder, in mg/100 g dw, was 34.1 ± 2.2 for iron, 1.9 ± 0.0 for zinc, and 2054.9 ± 11.5 for calcium. The iron, zinc, and calcium content of Cochlospermum tinctorium root powder, again in mg/100 g dw, were 26.8 ± 2.7, 0.9 ± 0.0, and 1,061.3 ± 11.5, respectively. The formulated complementary food supplement contained, in mg/100 g dw, 17.4 ± 1.1 of iron, 1.2 ± 0.1 of zinc, and 830.0 ± 0.2 of calcium. Except for copper, significant differences were found between the mineral content of the ingredients and the developed complementary food supplement (Table 2).

| Vitamins and antinutritional factors
The contents of vitamin A and vitamin C and the antinutritional factors of Adansonia digitata fruit pulp, Moringa oleifera leaf powder, Cochlospermum tinctorium root powder, and the complementary food supplement are presented in Table 3. Adansonia digitata fruit pulp had 372.7 ± 12.2 mg/100 g dw of vitamin C and 2,128.2 ± 44.5 mg eq AG/100 g dw of total phenols. Moringa oleifera leaf powder and Cochlospermum tinctorium root powder TA B L E 2 Dry matter (%), ash (%), and mineral (mg/100 g dw) contents of the selected food ingredients and the complementary food supplement

| Contribution of the 10 g complementary food supplement to the estimated average requirements
The study findings indicate that ten (10) grams of complementary food supplement covers 25% of the iron estimated average requirements for 6-to 12-month-old children and 58% and 42% of the iron estimated average requirements for 12-to 36-month-old children and 36-to 59-month-old children, respectively (Table 4). For zinc, the daily consumption of 10 g complementary food supplement would cover 5% of zinc estimated average requirements for children under 36 months old and less than 3% for 36-to 59-month-old children. The consumption of 10 g complementary food supplement per day would also cover 17% and 10% of the calcium estimated average requirements for 12-to 36-month-old and 36-to 59-month-old children, respectively. For vitamin C, the indicated consumption level of the complementary food supplement would contribute the total vitamin C estimated average requirements for 12-to 59-month-old children and 187% and 111% for 12-to 36-month-old and 36-to 59-month-old children, respectively.

| Acceptability of the complementary food supplement
Seventy-five percent (75%) of enrolled mothers were 18 to 30 years old and twenty-five percent (25%) more than 30 years old. Thirty percent (30%) of children were 6-12 months old, 45% were between 13 and 24 months old, and 25% were between 24 and 59 months

| Unenriched and enriched maize ogi porridge
The pH of maize ogi porridge decreased but not significantly from 3.86 ± 0.02 to 3.83 ± 0.04 after the enrichment with the complementary food supplement. In contrast, the degrees Brix and total color difference increased significantly after the enrichment from 5.9 ± 0.2 to 6.3 ± 0.2% and from 14.0 ± 0.1 to 39.0 ± 0.3, respectively.
The Bostwick flow rate (mm/30 s) after the enrichment of maize ogi porridge decreased significantly from 8.4 ± 0.4 to 6.9 ± 0.1.

| Nutritional potential of the selected food ingredients
The of Cochlospermum tinctorium root powder. The authors found a value of 70.1 mg/100 g dw for iron, which is higher than the value in this study (26.8 ± 2.7 mg/100 g dw). This discrepancy could be explained by the genetic background, the environmental conditions, the analytical methods, and the provenance of the sample (Chadare et al., 2009;Kayalto et al., 2013;Moyo et al., 2011;Muthai et al., 2017;Stadlmayr et al., 2013). Nevertheless, the iron content of Cochlospermum tinctorium root powder in this study still makes it suitable to be promoted as a local food fortificant to address the iron deficiency issue as suggested by Affonfere (2018). Cochlospermum tinctorium root powder can be successfully used in combination with Moringa oleifera leaf powder for food fortification to improve the iron status of children when Moringa oleifera leaf powder is not available and/or to mitigate its high cost, as Moringa oleifera leaf powder is fifteen (15) times more expensive than Cochlospermum tinctorium root powder. The vitamin C content of Adansonia digitata fruit (372.7 ± 12.2 mg/100 g dw) found in this study can contribute to achieving the vitamin C requirement for children under five years old. This is also important to improve the bioaccessibility of minerals, especially iron. The high calcium content of Moringa oleifera leaf powder and Cochlospermum tinctorium root powder is also beneficial to children. Calcium is important to calcification and dentition for children (Soetan et al., 2010).

| Affordability and nutritional benefits of the complementary food supplement: implication for alleviation of iron deficiency
The food ingredients of the complementary food supplement were locally available, and the processes to obtain these ingredients do not require any specific equipment. Local populations can therefore TA B L E 4 Contribution (%) of the 10 g complementary food supplement to the minerals and vitamin C estimated average requirements  The inhibitory effect of phytic acid on mineral absorption, especially calcium, iron, and zinc, is well known (Al Hasan et al., 2016;Hurrell & Egli, 2010), and phytate-mineral molar ratios were suggested to assess this effect (Al Hasan et al., 2016). The phytate/iron molar ratio of the complementary food supplement is 2.03:1, which is lower than the critical value (10-14) above which phytate is believed to strongly impair iron absorption (Saha et al., 1994). Accordingly, good iron bioavailability may be expected from the complementary food supplement. Furthermore, vitamin C is known to be a good promoter of iron absorption (Zimmermann et al., 2005). Due to the considerable vitamin C content of the complementary food supplement (vitamin C: iron molar ratio of 4.5:1), this will improve the iron bioavailability. Zimmermann et al. (2005) reported that a vitamin C: iron molar ratio ≥4:1 is needed for optimal iron absorption in food with high phytate content. The phytate/calcium molar ratio of the complementary food supplement is 0.03:1, which is lower than the critical upper value of 0.24 (Morris & Ellis, 1985), while the phytate/ zinc molar ratio is 34.99:1, which higher than the critical upper value of 10-15 (Morris & Ellis, 1985;Oberleas & Harland, 1981;Turnlund et al., 1984). These molar ratios suggest that the phytate content of the complementary food supplement might impair zinc absorption.
Apart from phytate, other antinutritional factors such as phenolic compounds could also impair mineral absorption. Hence, assessment of mineral bioavailability is recommended for the complementary food supplement and enriched porridges.
The overall acceptability underlined the generally positive responses from mothers (100% of the mothers liked both enriched porridges Regarding the children, the analyses of their nonverbal cues showed that most of them (87% for sorghum and 85% for maize porridges) liked the enriched porridges. The positive responses from children and their mothers regarding the acceptability of the enriched porridges will support the adoption of the developed product as acceptability is key to predict the intent to use a product (Barcenilla & Bastien, 2009;Février, 2011). Adoption here refers to an individual (or collective) decision to accept and use a product (Alexandre et al., 2018). Additionally, the low cost of the comple-

| CON CLUS ION
This study demonstrated the potential of local food ingredients in the formulation of a sensory acceptable complementary food supplement, which can be used by local populations to enhance their iron intake. The 10 g dry weight corresponding to 11.34 g wet weight daily consumption of the complementary food supplement covers 25 to 57% of iron estimated average requirements for children from 6 to 59 months old.

ACK N OWLED G M ENTS
This research was funded by the Regional Universities Forum for Capacity Building in Agriculture under BAOCHAIN project RU/2018/CARP+/01.

CO N FLI C T S O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L S TATEM ENTS
Ethical Review: This study does not involve any human or animal testing.
Informed Consent: Written informed consent of participants was obtained for the acceptability test after receiving complete information about the study in the local language.