Nutritional Quality, Mycotoxins and Antinutritional Factors in Quality Protein Maize-Based Supplementary Foods for Children in Tanzania

Most complementary foods used for children in Tanzania are low in energy and nutrient content. In addition, they may contain contaminants such as mycotoxins and also antinutritional factors. The aim of this study was to determine nutritional quality of quality protein maize-based supplementary foods and levels of mycotoxins (fumonisins, aflatoxins) and antinutritional factors (phytates, tannins). Three composite diets were prepared from quality protein maize namely; quality protein maize-soybeans; quality protein maize-soybeans-common beans and quality protein maize-soybeans-cowpeas. The fourth and fifth diets were prepared from plain quality protein maize and plain common maize. The formulations were made to meet the greatest amino acid scores and the desired amount of energy and protein according to the FAO/WHO (1985) recommendation for pre-school children. Concentrations of energy, protein, amino acid, aflatoxins, fumonisins, phytates and tannins were determined by standard methods. Quality protein maize-soybeans-common beans and quality protein maize-soybeans-cowpeas met RDA for both energy (360 kcal/100 g) and protein (16 g/100 g) for children aged 2-5 years. The amino acid scores for QPM-based diets were higher than the recommended scores (≥65%) for supporting optimal growth of children. Concentrations of fumonisin B1 and total fumonisin were 1687.82 and of 1717.16 μg/kg in quality protein maize and 1625.08 and 1745.22 μg/kg in plain common maize, respectively. These values were above the maximum tolerable limit of 1000 μg/kg recommended by the European commission. Efforts such as good agricultural practices and proper processing of food ingredients by sorting, dehulling and washing are recommended to reduce concentrations of fumonisins in maize grains.


Introduction
Adequate nutrition and health care during the first several years of life is fundamental for child growth, development and survival. Although the causes of malnutrition are many and diverse, inadequate intake of foods and essential nutrients is a major contributory factor. At about six months of age, breast milk supply of energy and other nutrients such as proteins, vitamins and minerals is no longer adequate to meet child's body needs (WHO, 2009). Therefore, a high energy and nutrient dense complementary foods must be provided to the child. In many developing countries, Tanzania inclusive, cereals and legumes are used as a basis for these complementary foods (Kulwa et al., 2015). These foods are usually prepared as thin gruels. As a result, their energy and nutrient density are low. Furthermore, cereals and legumes are susceptible to fungal contamination. One of these contaminants is mycotoxins. Mycotoxins are fungal secondary metabolites produced by toxigenic strains of fungi that contaminate crops before or after harvest. The most common mycotoxin producing fungi belongs to the three genera of fungi: Aspergillus, Penicillium and Fusarium (Frisvad et al., 2006). Ingestion of mycotoxins contaminated grains by animals and human beings has enormous public health significance, because these toxins are capable of causing diseases (Gnonlonfin et al., 2013). These toxins are nephrotoxic, immunotoxic, teratogenic and mutagenic. They are also capable of causing acute and chronic effects in man and animals ranging from disorders of the intestinal tract, central nervous, cardiovascular and pulmonary systems and death (Lombard, 2014). These mycotoxins have recently been associated with cancers and stunting in young children (IARC, 2016). The most toxic mycotoxins are aflatoxins, ochratoxin A, fumonisins, trichothecenes and zearalenone (Pitt, 2000).
Maize and beans are important food crops in Tanzania (Barreiro-Hurle, 2013 and ProFound, 2012). They are commonly used as ingredients in making complementary foods for children. Other cereals such as sorghum, rice, wheat and finger millet are rarely used. Apart from nuts, maize is more susceptible to mycotoxin contamination than any other crops. This is because corn production frequently encounters a period of drought and heat stress during flowering and kernel development. These weather conditions have been reported to increase mycotoxin contamination in maize than in other crops (Kebede et al., 2012). Maize contains about 45% carbohydrate, 5% protein and 2% fat in general (Enyisi et al., 2014). The protein content of beans ranges between 20-30% and 5% fat. Legumes are also rich in folate (50%), and minerals such as iron (4%), zinc (1%), and calcium (24%) (Messina, 1999). In addition to nutritional values of cereals and legumes, their use is limited due to presence of antinutritional factors. Phytic acid for example, reduces the availability of many minerals such as iron, zinc, calcium and magnesium (Asuquo & Etim, 2011). Ability of phytate to form complexes with these minerals makes the minerals not bioavailable. Tannins are known to bind proteins, including digestive enzymes leading to poor protein digestibility (McSweeney et al., 2001). Formation of protein complexes lowers protein digestibility, hence reducing food utilization and growth in children. It is therefore recommended that, when preparing complementary foods for children various methods should be used in inactivating and/or reducing these antinutritional factors. In Tanzania, some efforts have been made to determine nutrient content, levels of mycotoxins and antinutritional factors in cereal-based complimentary foods for children in plain or composite maize with legumes and/or nuts (Kimanya et al., 2014: Magoha et al., 2014). There is however, limited information documented about mycotoxin and antinutritional factors in quality protein maize (QPM)based formulations for children in Tanzania although QPM use has been growing steadily in the past decade. This study was carried out to determine the nutritional quality, mycotoxins (aflatoxin and fumonisin) and antinutritional factors (phytate and tannins concentrations) in QPM-based composite formulations used for supplementing children in Tanzania.

Materials and Methods Materials
Quality protein maize was purchased from Seliani Research Station in Arusha, Tanzania. Common maize (Zea mays), soybeans (Glycine max), common beans (Phaseolus vulgaris), cowpeas (Vigna unguiculata), edible vegetable oil and sugar were purchased from Morogoro Municipal central market.

Blend formulation
Formulations of high-protein-energy supplementary foods were made to meet the greatest amino acid score and the desired amount of energy and fat according to the FAO/WHO (1985) Codex Alimentarius guidelines for supplementary foods for infants and young children. The blend ratios of QPM, soybean, common beans, cowpeas and normal maize are shown in Table  1. QCS=Quality protein maize-Cowpeas-Soybean: QBC=Quality protein maize-common Beans -Soybean QQ=Quality protein maize; CM= Common Maize Food processing Separately, QPM, common maize (CM), soybeans, common beans and cowpeas were sorted to remove extraneous materials and pebbles and washed twice in distilled water. Then, QPM and CM were separately dehulled. Thereafter, each of these ingredients was separately milled into fine flour (mesh size 0.4 mm) using a commercial hammer mill (Intermek, Tanzania) to fine flour. Three formulations were developed; QPM-Soybeans (QS), QPM-soybeans-common beans (QSB) and QPM-soybeans-cowpeas (QSC). The fourth product was made from QPM alone (QQ) and fifth products from common maize alone (CM). Each food product was conditioned to 22% moisture content and 5% vegetable oil and allowed to equilibrate for 30 minutes. The food mixtures were separately extruded using a commercial twin-screw extruder (Model JS 60 D, Qitong Chemical Industry Equipment Co. Ltd, Yantai, China) with two electrically heated zones. The following extrusion conditions were adopted: Temperatures 130 o C (Zone 1) and 122 o C (Zone 2), main motor speed was set at 10.48 rpm and feeder speed at 10.26 rpm. Desired barrel temperature was maintained by circulating cold water. Temperature was controlled by inbuilt thermostat and a temperature control unit. The extruder was cleaned after each run with moist maize flour. After extrusion, the extrudates were allowed to cool and dry at room temperature, thereafter milled, fortified and packaged in polyethylene packets ready for laboratory analysis.

Chemical Assays
Crude protein, energy and amino acid determination Crude protein was determined according to AOAC (1995). Carbohydrate content was calculated by difference. Energy values (kcal) were calculated by using Atwater conversion factors 4, 9 and 4 for each gram of protein, fat and carbohydrate, respectively (AOAC, 1995). Amino acid concentrations (except tryptophan) were determined by a high performance liquid chromatography using the Waters Pico-Tag method (Cohen et al., 1989). For all amino acids except methionine, cysteine and tryptophan, food samples were hydrolysed in 6 N HCl. The methionine and cysteine in foods were oxidised by performic acid to methionine sulfone and cysteic acid prior to hydrolysis by 6 N HCl. All amino acids (except tryptophan) were derivatised by phenyl isothiocayanate and detected at 254 nm. Tryptophan was analysed by ion exchange chromatographic method as described in the AOAC (1995) method 988.15. The protein in the food was hydrolysed under vacuum with 4.2 N NaOH. After pH adjustment and clarification, tryptophan was separated by ion exchange chromatography (DC5A cation exchange resin) with measurement of the ninhydrinchromophore.

Amino acid score
The amino acid score was calculated using the ratio of a gram of the limiting amino acid of test diet to the same amount of the corresponding amino acid in the reference protein multiplied by 100. The reference proteins suggested by FAO/WHO/UNU (1985) for children aged 2-5 years was used for calculating amino acid score.

Aflatoxins and fumonisins determination
Aflatoxin concentration was determined according to method described by Kimanya et al. (2008). Determination of fumonisins B1 and fumonisins B2 was carried out using HPLC method described by Samapundo et al. (2006) and Kimanya et al. (2008). Phytic acid concentration was determined by the method described by Wheeler and Ferrel (1971). Tannin was determined following the method described by Porter et al. (1986).

Statistical analysis
All data (except for amino acids), were subjected to analysis of variance (ANOVA) using Genstat (1998, version 4). For amino acids, mean values were computed. The concentrations of mycotoxin and antinutritional factors in the composite products were compared by using the Turkeys Least Significant Difference (LSD) test when difference existed. Differences were considered significant at p≤0.05.

Results and Discussion Energy, protein content and amino acid profile of QPM-based composite foods
Chemical assays showed an increase in protein content of the composite diets blended with soybeans. The energy and protein density of the diets were 386 kcal and 17 g per 100 g of QSB, 389 kcal and 16 g per 100 g of QSC, 381 kcal and 16 g per 100 g of QS,387 kcal and 6 g per 100 g of CM. Comparing with FAO/WHO reference, diets QSB and QSC met both energy (360 kcal) and protein content (16 g) per 100 g of edible food required for children aged 2-5 years. This could partly be attributed to the higher contents of protein supplemented to QPM blended flour. These findings were in line with those reported by other investigators ( Amino acid profiles of the experimental and control diets are presented in Table 2. Amino acid composition of the composite diets ranged between 15 mg/100g for tryptophan (QQ diet) and 109 g/100g for leucine (CM diet). Lysine and tryptophan contents of the composite diets increased with soybean supplementation. Lysine content in QPM-based diets ranged from 67 to 74 mg/100 g protein in QS and QSB, respectively. Tryptophan concentration ranged from 16 to 18 mg/100 g protein in QS and QSB, respectively. Lysine and tryptophan contents of QSB and QSC were higher than the levels in reference protein for children aged 2-5 years (58 mg/g Lys and 11 mg/g Trp) (FAO/WHO/UNU, 1985). Plain common maize diet (CM) contained lower concentration of lysine and tryptophan than the recommended levels of Lys (58 mg/g) and Trp (11 mg/g) protein, for children 2-5 years. This study revealed that, histidine, threonine, valine, leucine, lysine, tryptophan, sulphur containing amino acids (methionine and cysteine) and aromatic amino acids (phenylalanine and tyrosine) were present in adequate amounts in the QPM-based diets when compared with the recommended levels for children 2-5 years by (FAO/WHO/UNU 2002). Amino acid that is in short supply is referred to as the most limiting amino acid (Häffner et al., 2003). Results of amino acid score in the test diets indicated that lysine was the most limiting amino acids in all the diets.  According to Gheysens (2015), concentrations of mycotoxins in maize grains were fumonisn B1 (0.00-5461.00 μg/kg), fumonisin B2 (0.00-1756.80 μg/kg) and total fumonisins (0.00-6761.00 μg/kg). The maize used for their study were just crushed prior to analysis of mycotoxins unlike the maize grains used in our study which were sorted, winnowed, washed, dehulled, milled and extruded before analysis. These processes were missing in the Gheysens (2015) study. Magoha et al. (2014) reported that 68% of the flour samples collected from Northern Tanzania were contaminated with total fumonisin concentration above the maximum tolerable limit (MTL). A study by (Kumi et al., 2014) in weanmix observed that, mean fumonisin contamination was 4.6 μg/kg in the composite flour. Furthermore, 58.3% of the weanmix samples were contaminated with fumonisin concentration above the U.S. FDA limit of 4 μg/kg. Chronic mycotoxin exposure has major effects on nutritional status in human. It suppresses body's immunity and nutritional status. Aflatoxin B1 is known to be acutely toxic and a cause of liver cancer in humans (Shephard, 2008). Aflatoxin exposure has also been associated with an increased risk for liver cirrhosis (Kuniholm et al., 2008). Fumonisin and aflatoxin exposure early in life has been associated with impaired growth, particularly stunting in Tanzania (Kimanya 2010) and West Africa (Gong et al., 2003).

Antinutritional factors of the formulated diets
Concentrations of phytate and tannin in the composite foods are presented in Table 3. Concentrations of phytic acid among the food products were significantly different (p<0.05), increasing with the level of legume substitution (16.6 to 24.6mg/100g). The concentrations of phytates in the supplementary foods studied were lower than 25 mg/100g, the amount considered lethal to health (Nagel, 2010). Similarly, tannin levels increased with legume supplementation. Tannins concentrations were very low (0.12-3.5 mg/100g) while the reported lethal dose is 90 mg/100g (Ifie & Emeruwa, 2011). This indicated that, the concentrations of phytates and tannins in the composite diets were of acceptable safe levels and the foods were therefore safe for human consumption.  (Anton et al. (2009) reported that, extrusion cooking reduced phytic acid inhibitor levels to nearly 50% in maize-common bean blend. Concentrations of phytates and tannins observed in this study posed no health risk to the consumers. Commission. Based on these results, it is recommended that efforts must be made to reduce the levels of fumonisins in maize grain. This can be achieved through good agricultural practices and proper processing practices of food ingredients such as sorting, dehulling and washing when making supplementary foods for children.

Conflict of interests
The authors declare that they have no conflict of interest.