Nutritional and antinutritional evaluation of complementary foods formulated from maize, pea, and anchote flours.

Abstract This study was aimed to evaluate nutritional and antinutritional contents of complementary foods from locally available and affordable raw materials (maize, pea, and anchote) grown in Western Ethiopia. The six formulated complementary diets analyzed for their proximate, mineral, and antinutritional continents were compared with Codex standards. The mineral ratios and molar ratios of the formulated diets were also evaluated and compared with each standard values. Six formulations were generated by d‐optimal mixture design. The formulated ingredient ranges 45%–61% maize, 23%–31% pea, and 14%–28% anchote. Design‐Expert® 6 (Stat‐Ease) was used to constrain the three components. The formulated diets ranged from 14.92% to 20.99%, 5.95% to 9.94%, 2.75% to 3.41%, and 59.10% to 66.22% of protein, fat, fiber, and utilizable carbohydrate, respectively. Mineral contents (mg/100 g) of the formulated diet ranged from 225.45 to 261.32, 11.48 to 12.61, 2.73 to 3.00, 357.92 to 391.13, 298.55 to 332.63, 252.00 to 278.01, and 44.26 to 51.56 for calcium, iron, zinc, phosphorous, potassium, sodium and magnesium, respectively. The proximate and mineral contents of the formulated diet 5 meet the Codex standards, except the fat contents of the complementary food standards. The molar ratios of the formulated diets in this study were below standard reference and which show the high mineral bioavailability in all the formulated diets. The results of the study revealed that the formulated diets contain very low antinutritional factors and high mineral bioavailability. The paper's findings show that the complementary food formulated from maize, pea, and anchote flours particularly diet 5 may be suitable to alleviate protein energy malnutrition and it can be used as a substitute for the expensive commercial complementary food.


| Description of sampling site
Maize, pea, and anchote samples were collected from Nekemte market. Nekemte, the capital town of East Wollega Zone is located 322 km away from capital city of the country, in the western part of the country. In East Wollega Zone, agriculture (crop cultivation and rearing of livestock) is the leading economic activity in the district.

| Preliminary survey
To collect data from the respondent interview, semistructured questionnaire was prepared. Twenty-two respondents were selected purposefully. Among them, twelve respondents were mothers who have complementary age children during the interview, six respondents were health extension and four respondents were agricultural expertise who possesses knowledge of complementary food production, marketing, and consumption of locally available complementary foods. The central theme of the interview was to gather prior information about production, handling, processing, and utilization of complementary food. The initial survey data lead the experimenter to have basic information on the type and production of locally available complementary food produced and to fix the commonly available complementary food for sample selection. Based on the respondent response, maize, pea, and anchote are the commonly used complementary food locally available in East Wollega Zone, Ethiopia. In addition, wheat and teff are also used for complementary food formulation. Based on this information, we have selected known variety of maize, pea, and anchote for the formulation of locally available foods in East Wollega Zone, Ethiopia.

| Sample collection and preparation
Maize (BH66 variety, 2 kg), pea (karse variety, 2 kg), and anchote (dime, 6 kg) samples were purchased from the local market, Nekemte, Ethiopia. The collected samples were coded, packed, and prepared at Wollega University research laboratories of Food Technology department. The maize and pea samples were sorted and sun-dried separately and then oven-dried at 45°C. Anchote tuber was washed by distilled water, was sliced to uniform thickness of 5 mm using a stainless-steel knife, and was sundried. All the dried samples were milled separately into a fine powder (0.425 mm sieve size). The moisture content of the samples was determined immediately after milled into fine powder. Finally, the powder was packed into airtight polyethylene plastic bag and was stored in a desiccator until required for further analysis. All chemicals used were of analytical grades. Table 1 shows the d-optimal mixture design obtained from generated experimental design and the embarrassed area is shown in Table 2. Three mixtures were made from treatment including the following: 14 < anchote < 28, 45 < maize < 61, and 23 < pea < 31 (anchote + maize + pea = 100%). Therefore, from the treatment combinations, six samples were generated. A blend of these ingredients was therefore expected to give complementary foods of very balanced nutritional value.

| Determination of moisture content
The proximate composition analysis including moisture content, crude protein, crude fat, crude ash, and crude fiber was determined according to approved AOAC (2000) method. Utilizable carbohydrate content was calculated by difference, that is, 100 − (% crude protein + % crude fiber + % total ash + % crude fat). The gross energy content was determined by calculation from fat, carbohydrate, and protein contents using conversion factors; 4 kcal/g for protein, 9 kcal/g for fat, and 4 kcal/g for carbohydrates (Guyot, Rochette, & Treche, 2007).

| Determination of mineral content
The minerals were determined according to the standard method of AOAC (2000). Calcium, iron, and zinc were determined by using atomic absorption spectrophotometer (AAS), while sodium and potassium contents were determined using flame photometer (Jenway, PF 7). Phosphorus was determined by the colorimetric method using ammonium molybdate (AOAC, 1984).

| Determination of mineral ratios
The mineral ratios are often more important than individual mineral levels themselves because they are useful in determining nutritional interrelationships and also provide information regarding the many possible factors that may be represented by a disruption of their relationships such as disease states, physiological and developmental factors, and the effects of diets (Hoskin & Ireland, 2000). The mineral ratio was calculated by dividing the first mineral level to the second mineral level (Jacob, Etong, & Tijjani, 2015).

| Determination of phytate content
Phytate was determined by the method described by Vaintraub and Lapteva (1988). Oxalate was analyzed using the method originally used by Ukpabi and Ejidoh (1989). Tannin content was determined according to the method described by Maxson and Rooney (1972).

| Determination of molar ratio of antinutrients to minerals
The molar ratio was predicted by dividing the mole of antinutrient to the mole of minerals (Norhaizan & Norfaizadatul, 2009).

| Statistical analysis
Mixture design using Design-Expert ® 6 (Stat-Ease) was used to constrain three components. The completely randomized design was employed with two replicates. SPSS version 20.0 for windows was used to perform all the statistical analyses. One-way analysis of variance (ANOVA) was used to evaluate the data. Duncan's multiple range test was used to separate means, and the result was reported as a mean ± standard error (SE). A p-value of .05 or less was considered as the statistically significant difference. 12.20% for maize, pea, and anchote flours, respectively. Crude protein content of pea flour was significantly (p < .05) higher than maize and anchote flours. Akingbala, Akinwande, and Uzo-Peters (2003) reported that pea contains appreciable protein, and this study also revealed that pea seeds are a good source of protein for potential for complementary food formulation. In addition, consumption of the seeds should be encouraged to mitigate protein energy malnutrition in the country.

| Proximate composition of the ingredients and formulated diets
The crude fat contents of the ingredients were 12.86%, 2.59%, and 1.93% for maize, pea, and anchote flours, respectively. Maize flour was significantly (p < .05) higher in crude fat content followed by pea and anchote flours. The ash stuffing of the component was 2.54%, 2.56%, and 3.87% for maize, pea, and anchote flours, respectively. Anchote tuber contained fairly high ash content which is an indication that anchote would provide essential minerals needed for body development. Anchote flour was also significantly (p < .05) higher in crude ash content than maize and pea flours. Crude fiber contents of the ingredients were 2.51%, 1.92%, and 7.39% for maize, pea, and anchote flours, respectively.
Anchote flour was significantly (p < .05) higher in crude fiber content and followed by maize and pea flours. This finding revealed that anchote tuber is considered as a main source of crude fiber.
Utilizable carbohydrate contents of the ingredients were 58.81%, 61.86%, and 67.69% for maize, pea, and anchote flours, respectively. The gross energy contents (kcal/100 g) of the ingredients were 426.14, 374.91, and 336.93 for maize, pea, and anchote flours, respectively. Gross energy contents of maize flour were higher than pea and anchote flours. This indicates that maize flour could be a major source of energy.
The proximate composition of the formulated diet is presented in Table 4. The range of moisture content of the six formulated diets was from 4.78% to 5.31%. Diet 3 having the blend of 30.6% pea, 24.4% anchote, and 45.0% maize were the highest in crude fiber while the lowest moisture contents were observed in diet 2 with the blend of 13.5% anchote, 28.7% pea, and 57.8% maize (Table 4). When the proportion of maize decreased and increased with anchote flour blend, the moisture content was increased. In this finding, the formulated diet 6, diet 5, and diet 2 convene the recommended moisture content (<5%) by CODEX CAC/GL (1991) (   The mineral contents of the formulated diets are presented in Iron, zinc, phosphorus, magnesium, and calcium have been identified as the problem nutrients from 6 months of age and must be supplemented with the addition of complementary food (Bjelakovic, Nikolova, Gluud, Simonetti, & Gluud, 2007). The mineral content of the current study was almost within the range recommended by CODEX CAC/GL (1991) except iron and potassium contents (Table 6).

| Mineral ratios of the formulated diets
The effectiveness of minerals in the diets is influenced by min- ratio of the formulated complementary diets in this investigation revealed that consumption of these complementary diets would help to prevent hypertension and might lower blood pressure and may also be suitable for children who have the risk of high blood pressure.
The calcium-phosphorous (Ca:P) ratios of the six formulated complementary diets ranged from 0.606 to 0.684. The recommended Ca/P ratio should be >0.5 (Jacob et al., 2015).
Furthermore, food is considered as good if Ca/P ratio is >1 and poor if this ratio is <0.5 (Alinnor & Oze, 2011). Chandran, Nivedhini, and Parimelazhagan (2013) also reported that the Ca/P ratio must be close to 1 for a good Ca and P intestinal utilization. A higher calcium-phosphorous (Ca/P) levels in foods are required for favorable calcium absorption in the intestine for bone formation (Adeyeye, Orisakeye, & Oyarekua, 2012). According to Adeoti et al. (2013), diets rich in protein and phosphorus may promote the loss of calcium in the urine. The Ca/P ratio in this study indicates that the formulated complementary diets would help calcium absorption in the body. The high Ca/P ratio observed in this study is of nutritional benefit, particularly for children and the aged who need higher intakes of calcium and phosphorus for bone formation and maintenance. It is well known that diets with a high value of Ca/P ratio are considered good, particularly for growing children who require a high intake of calcium and phosphorus for bone and teeth formation (Oluwole, Adeoti, & Ariyo, 2013).
The calcium-potassium (Ca/K) ratios of the six formulated complementary diets ranged from 0.716 to 0.815. The Ca/K ratio is called the thyroid ratio because calcium and potassium play a vital role in regulating thyroid activity (Olagbemide, Ojiezeh, & Adarabioyo, 2016). Low Ca/K ratio would indicate an elevation of thyroid expression (Watts, 2010 The iron-zinc (Fe/Zn) ratios of the six formulated complementary diets ranged from 4.203 to 4.217. Pérès, Bureau, Neuville, Arhan, and Bouglé (2001) reported that iron did not impair zinc absorption up to an iron: zinc ratio of 2:1; then a dose-dependent effect was observed up to a ratio of 5:1; when the ratio was increased from 5:1 to 10:1, no further inhibition of zinc occurred. Based on this report, one can conclude that the iron present in the formulated complimentary diets did not impair zinc absorption.

| Molar ratios and bioavailability of the formulated complementary diets
Bioavailability is the proportion of the total amount of mineral element that is potentially absorbable in a metabolically active form (Šimić et al., 2009). The calculated values of the molar ratios were also compared with the reported critical toxicity values for these ratios.  (Siegenberg et al., 1991). This result indicated that all the formulated diets contain the phytate:iron molar ratios of less than the critical value, this implies the high bioavailability and absorption of iron.
The molar ratios of phytate to zinc of the formulated diets varied from 2.30 to 2.52. The importance of foodstuffs as a source of dietary zinc depends on both the total zinc content and the level of other constituents in the diet that affect zinc bioavailability. The bioavailability of dietary zinc might be reduced by phytate (Bhandari & Kawabata, 2004). Hence, the Phy:Zn molar ratio is considered a better indicator of zinc bioavailability than total dietary phytate levels alone (Woldegiorgis et al., 2015). Foods with a molar ratio of Phy:Zn < 10 showed adequate availability of zinc, and there will be a problem encountered when the value is >15. Phy:Zn molar ratios > 15 is an indication of poor zinc bioavailability (Morris & Ellis, 1989