Materials
Raw sorghum, millet, maize, rice, water yam, sweet potato and cocoyam were bought from Agbogloshie market in Accra, Ghana and transported to the processing laboratory of CSIR-Food Research Institute, Accra for processing into flour.
Methods
Sample preparation
Cocoyam flour preparation: Matured and healthy cocoyam cormels were peeled with sharp stainless steel knife. Peeled cocoyam was washed three times to remove dirt and impurities. The washed peeled cocoyam was sliced into small pieces using the stainless steel knife for easy drying. It was blanched by steeping in hot water for 2-3 minutes and solar dried on thinly cleaned black plastic sheet till it became firm and brittle. Solar dried cocoyam slices were milled using hammer mill and milled flour sifted using sieve to remove lumps to ensure free-flow flour with even particles was obtained. The flour was packaged into sacs lined with moisture-proof polythene and sealed airtight using an electric impulse sealer till analysis was carried out (Tortoe et al. 2014). Sweet potato flour and water yam flour were prepared the same way as the cocoyam flour was prepared.
Maize flour preparation: All the three cereal flour samples were prepared using a method by Addo et al. 2015. Mold-free and weevil-free maize was selected. It was poured onto a tray for sorting. Foreign materials such as stones and insect infested maize were removed through winnowing. The maize was dehulled after sorting. The dehulled maize was solar dried on cleaned black plastic polythene sheet until moisture content was about 11 %. The dried dehulled maize was milled using hammer mill. The milled flour was sifted using a 250 microns sieve to remove lumps so as to obtain a free-flow flour. The maize flour was then put in sacs lined with moisture-proof polythene and sealed with air-tightened electric impulse sealer. The rice flour was prepared the same way as the sorghum flour.
Millet flour preparation: Mold-free and weevil-free millet was selected. Millet grains was poured into a plastic bowl containing enough water. Suspended water millet grain was decanted little at a time using calabash to ensure that millet grains are free of stones and impurities. This process was repeated several times till all stones and impurities are removed from the millet grains. Cleaned millet grains was solar dried by spreading it on a thin black plastic sheet. Millet grains was dehulled and solar dried the second time till moisture content of 11 %. The dehulled dried millet was milled into flour using hammer mill. The flour was sifted using a sieve of mesh size of 250 microns to remove all lumps.
Sorghum flour preparation: the variety of sorghum processed was the Beiko peleg which is suitable for the preparation of local food (tuo zaafi). Clean and mold-free sorghum grains was selected for the flour production. Sorghum grains was washed with clean water to remove dirt, stones and other foreign materials. Grain was solar dried by spreading it on a clean black polythene sheet till moisture content was 11 %. Grains was dehulled and solar dried for the second time to remove most of the moisture content. It was milled using the hammer mill into flour and sifted using sieve of mesh size of 250 microns to remove lumps.
Chemical analyses
Trace metal content determination
The method used by (Ofori et al. 2016) was used for the trace metal analyses. The dry ashing method was used for the Atomic Absorption Spectrometry (AAS) analysis (AOAC 2005). All glass ware was washed with 1 % nitric acid followed by demineralized water. Three grams of each sample was weighed into a platinum crucible. The crucible and test sample was placed in muffle furnace at a temperature of 550 °C for 8 hours. The crucible with ash was put in desiccator to cool. Five (5) mL of nitric acid of mass fraction not less than 65 %, having a density of approximately ρ (HNO3) = 1400 mg.mL-1 was added, ensuring that all the ash came into contact with the acid and the resultant solution heated on hot plate until the ash dissolved. Ten (10) mL of 0.1 mol.L-1 nitric acid was added and filtered into 50 mL volumetric flask. The resultant solution was topped up to the mark with 0.1 mol.L-1 nitric acid. Blank solution was treated similar as the sample. Buck Scientific 210VGP Flame Atomic Absorption Spectrophotometer (Buck Scientific, Inc. East Norwalk, USA) was used to read the absorbance values at appropriate wavelength of the interested metal in sample solution. Cathode lamps used were As (wavelength 193.7 nm, lamp current 4.0 mA), Cu (wavelength 324.8 nm, lamp current 1.5 mA), Fe (wavelength 248.3 nm, lamp current 7.0 mA), Hg (wavelength 253.7 nm, lamp current 0.7 mA), Pb (wavelength 217.0 nm, lamp current 3.0 mA), and Zn (wavelength 213.9 nm, lamp current 2.0 mA). The metal content of the sample was derived from calibration graph made up of a minimum of three standards.
Aflatoxins determination
The extraction procedure used for the determination of aflatoxins was by the Stroka and Anklam (1991). A test portion (50 g) was extracted with 200 ml methanol/water solvent solution containing 5 g of sodium chloride. The sample extract was filtered, diluted with phosphate buffered saline to a specified solvent concentration and applied to the immunoaffinity column (R-Biopharm Rhone Ltd. Easi-Extract Aflatoxin) containing antibodies specific for aflatoxins B1, B2, G1 and G2. Aflatoxins were eluted from the immunoaffinity columns with neat methanol. The Aflatoxin level was quantified by reverse-phase high performance liquid chromatography (RP-HPLC) with post column derivatisation (PCD) involving bromination. The PCD was achieved with pyrimidinum hydrobromide perbromide (PBPB) followed by fluorescence detection. HPLC system used for analyses was from Waters Associates (Milford, MA, USA) and included Waters 1525 Binary HPLC pump, Waters 2707 Autosampler, Waters Model 1500 Column Heater, Waters 2475 Multi λ Fluorescence Detector and Breeze 2 software. Separation of the aflatoxin was carried out on a Spherisorb S5 ODS-1 column of dimensions 25x4.6 mm packed with 5 μm particles (phase separation In., Norwalk, USA) maintained at 35 °C. The HPLC mobile-phase flow rate was 10 ml/min and post column bromine derivatisation of Aflatoxin B1, and G1 was achieved by PBPB dissolved in 500 ml of demineralised water pumped at a flow rate of 1.0 ml/min using Elder precision metering pump (Elder laboratories Inc., Sam Carlos, USA). The excitation and emission wave length used were 360 nm and 440 nm respectively. The Aflatoxin were identified by means of their retention times, and quantification was performed by comparing the peak areas of the samples to those of the standards prepared from pure aflatoxins standard (obtained from R. Biopharm) solutions under identified conditions.
Quality control of results
Samples were handled carefully to avoid contamination as part of measure to ensure reliability of results. The recovery test of the total analytical procedures was also carried out for the metals analyzed in the selected samples by spiking analyzed samples with aliquots of metal standards and then reanalyzed the samples. Acceptable recovery ranges of 94 ± 1 to 95 ± 1 %, 96 ± 1 to 97 ± 1 %, 93 ± 1 to 95 ± 1 %, 93 ± 1 to 94 ± 1 %, 95 ± 1 to 96 ± 1 %, 95 ± 1 to 97 ± 1 % were obtained for As, Cu, Fe, Hg, Pb and Zn respectively.
Data analysis
The standard deviations on mean values of duplicate samples were analyzed using Statistical Package for Social Scientist (SPSS 2013), version 21. Analyses of Variance (ANOVA), Duncan test was used to compare the means.