2.2. Determination of Proximate Composition

Determination of proximate composition (moisture, ash, crude protein, crude fat, and crude fiber) of maize flour, oyster mushroom flour, and the blended maize-mushroom composite flour was performed using standard official analytical methods AOAC [17].

The Kjeldahl method AOAC [17] was used to analyze crude protein content in each sample, according to Chang and Zhang [18]. Firstly, a mixture of K₂SO₄ (10 g), CuSO₄ (1 g), and selenium powder (0.1 g) was prepared and used as a Kjeltec catalyst. Afterwards, 10 ml of concentrated H₂SO₄ and Kjeltec catalyst was added in 1 g of each sample for digestion. The mixture was heated at 420°C for 2 hours to allow the digestion of the sample. About 10 ml of 0.5 NaOH was added in 10 ml of digested samples to provide a basic condition. Following the reaction, NH₃ was collected as NH₄OH in a conical flask containing 20 ml of 4% boric acid and a drop of modified methyl red as an indicator. Thereafter, the distillate was titrated against 0.1 N HCL solution until the endpoint was obtained. The percentage nitrogen in the sample was determined using the following expression:

where V₁ is the titer for the sample (ml), V₂ is the titer for blank (ml), V is the volume of diluted digest taken for distillation (10 ml), N is the normality of HCL solution, f is the factor for standard HCL solution, 0.014 is the milliequivalent weight of nitrogen, and S is the weight of the sample taken (g).

The amount of crude protein in the sample was determined by multiplying the percentage nitrogen with 6.25 as a protein conversion factor using the following expression:

The determination of crude fat in the sample was performed using standard procedure AOAC [17] with petroleum ether as an extracting solvent, as described by Nielsen and Carpenter [19]. Extraction of fat was done by placing 5 g of each sample of maize flour, oyster mushroom flour, and the blended maize-mushroom blended flours in the extraction thimble of Soxhlet apparatus (FOSS-Soxtec 2055, Denmark), followed by immersing the thimble inside the extraction containing 55 ml of petroleum ether. The extraction thimble with a fat-containing sample was heated to 60°C for 6 hours. Thereafter, solvent removal was done with a vacuum rotary evaporator's aid at 40°C, while fat drying was done in the dry oven at 70°C for 30 minutes. The amount of crude fat was obtained by subtracting an empty flask from the weight of the flask containing dried fat. The percentage of crude fat was obtained using the following expression:

where W₁ is the weight of the sample before extraction and W₂ is the weight of the sample after extraction.

The amount of crude fiber in each sample was determined using standard procedures of AOAC [17] described by Nielsen [20]. About 2 g of each sample was added to a mixture of 200 ml of 1.25% H₂SO₄ and 0.31 N NaOH, boiled for 30 minutes, and washed with ethanol and petroleum ether twice. The residues obtained were then placed in clean, dry weighed crucibles and dried overnight at 100°C inside the moisture extraction oven. Thereafter, the crucibles were heated in a muffle furnace at 600°C for 6 hours, cooled, and weighed again. The weight differences of the crucibles were noted as crude fiber and calculated as percentage crude fiber as expressed as follows:

where W₁ is the weight of the sample before heating, W₂ is the weight of the sample after heating, and W₃ is the weight of the original sample.

The ash content in each sample was determined using the AOAC [17] standard procedures described by Harris and Marshall [21]. The carbolite muffle furnace was used to heat the clean empty crucibles at 600°C for 1 h. The empty crucibles were weighed after cooling in a desiccator. The sample (2 g of each) was then placed in the crucibles, and their weight recorded, followed by burning in the muffle furnace at 550°C for 6 hours. The burnt crucibles containing samples were cooled in the desiccator and weighed again. The percentage of ash content was determined by using the following expression:

where W₁ is the weight of empty crucible, W₂ is the weight of the sample, and W₃ is the weight of the heated sample and the crucible.

Determination of moisture content in each sample was performed using the AOAC [17] standard procedures described by Bradley [22]. About 2 g of each sample was weighed into a dried moisture dish and placed in the moisture extraction oven (Wagtech, Germany) at 105°C for 5 hours. Thereafter, the dried samples were cooled in a desiccator and weighed again. This process was repeated thoroughly until the constant weight was attained. The percentage of moisture content of samples was calculated using the following expression:

where W₁ is the weight of the fresh sample (g) and W₂ is the weight of the dry sample (g).

The percentage of moisture content (% MC) was used to obtain the dry matter content of the sample using the following equation:

The content of carbohydrate in each sample was determined by the method described by Nielsen [20]. The percentage of carbohydrate was obtained by subtracting 100 from the summation of crude protein, crude fat, crude fiber, ash, and moisture content.

The energy value was determined using the method described by Farzana and Mohajan [23]. For each blended flour sample, the energy value was obtained as the summation of crude protein, crude fat, and carbohydrate and their respective physiological values (Atwater's conversion factors) of 4, 9, and 4 calories.

The mineral (iron, zinc, calcium, and potassium) contents [24] of control maize flour and complementary flour blends were determined using atomic absorption spectrophotometer (AAS) (Thermo Scientific® iCE 3500, Waltham, USA). The AOAC [17] standard procedures were followed as described by Yeung et al. [25]. The wavelengths used in reading the absorbance of cations in the AAS were 248.3 nm for iron (Fe), 213.9 nm for zinc (Zn), and 422.7 nm for calcium. Potassium was determined using an air-liquefied petroleum gas flame on a flame photometer. The calculation of mineral content (mg/100 g) in the sample was done, as shown as follows:

where R is the absorbance reading in ppm, 100 is the volume of sample made, D.F is the dilution factor, 1000 is the conversion factor to mg/100 g, and S is the sample weight.