PARTO: LEGRADO: CESAREA:

The colorimetric method was used to determine the phytate content of each sample (Onwuka, 2005).

Two grams (2 g) of each sample was mixed with 0.2 N HCl to form 1.25 w/v mixture. The mixture was shaken and allowed to stand for 30 minutes at room temperature. It was filtered with whatman no 42 filter paper to obtain the extract used in the analysis.

55 An aliquot (0.5 ml) of the extract was mixed with lml of Fenn solution in a test tube. It was heated in water GFL water bath (1083, Germany) for 30 minutes and then cooled in ice for 15 minutes. It was then allowed to attain room temperature (32^oC). It was later treated with 2ml of 2,2-Bipyridine solution, mixed well and its absorbance was read in a Jenway Digital Spectrophotometer (6051, UK) at 510 ɲm, meanwhile a standard phytate solution (sodium phytate) was prepared and diluted to 0.5 mg/ml. One (1) ml of the standard solution was treated as described above and its absorbance was also read. The percentage phytate content was calculated with the formula given below:

Percentage (%) phytate = 100 x au x C x Vt W as 100 Va

Where: W = weight of sample au = absorbance of sample

as = absorbance of standard solution Vt = total extract volume

Va = volume of extract analyzed

3.7.0 Proximate Analysis

Analyses of the proximate contents of the leaves, petioles, cormels and roots of five varieties of the species of C. esculenta were carried out according to the methods described by Onwuka (2005) with the exception of fat determination.The methods are described below:

3.7.1 Moisture Content Determination

56 The dishes were washed thoroughly and dried in the oven. They were latter put inside the dessicator to cool. After which they were weighed. Sample was put into the weighed dish and weight taken. The sample was dried in the oven at 70^oC for 2 hrs and at 105^oC for the next 4 hrs. The sample was cooled in the dessicator and the dry weight of sample plus dish taken. The moisture content was calculated as follows:

Percentage (%) moisture = W2 – W3 x 100 W2 - W1 1 Where:-

W1 = Initial weight of empty crucible

W2 = Weight of crucible + sample before drying W3 = Final weight of crucible + sample after drying

3.7.2 Ash Content Determination

Five grams (5 g) of finely ground dry sample was weighed into a tarred silica crucible. The sample was charred on a heater inside a fume cupboard, to dry off most of the smoke. The sample was transferred into a pre-heated muffle furnace at 550^oC. It was left at this temperature for 2 hours. After which it was cooled in a dessicator and re-weighed.

Percentage (%) Ash = Weight of ash x 100 Weight of original sample 1

57 = W3 –W1 x 100

W2 –W1 1 Where:-

W1 =Weight of empty crucible

W2 =Weight of crucible +sample before drying W3 = Weight of crucible + ash

3.7.3 Crude Fibre Content Determination

Water reflux was boiled for 30 minutes with 200 ml of a solution containing 1.25 g of H2SO4 per 100 ml of solution. The solution was filtered through two folds of cheese cloth on a fluted funnel. The residue was washed with boiling water until there was no longer acid. The residue was transferred to a beaker and boiled for 30 minute with 200 ml of a solution containing 1.25 g of carbonate–free NaOH per 100 ml. The final residue was filtered through a thin but closed pad of washed and ignited asbestos in a Gooch crucible. It was dried in an electric oven and weighed. It was finally incinerated, cooled and weighed.

The loss in weight after incineration x 100 was the percentage of crude fibre.

3.7.4 Crude Protein Content Determination

A measured weight (2 g) of sample was weighed into a 250 ml beaker. After which 75 ml of hot water was added and brought to boil. It was stirred vigorously and added 25 ml of 6% copper sulphate solution. It was again brought to boil, stirred vigorously and added 25 ml of the 1.25% sodium

58 hydroxide solution. The mixture was stirred vigorously, removed from the flame and allowed to settle. It was filtered through a 15 cm, No. 4 Whatman paper. The precipitate was cleaned from the sides of the beaker. The paper was washed free from sulphate with very hot water 6 times. It was allowed to drain well, and then transferred to a kjeldahl flask containing about 10 g anhydrous sodium hydroxide and a trace of selenium. Thirty milliliters (30 ml) of conc. H2SO4 was added, nitrogen content determined and hence the protein content of the sample.

Percentage (%) protein = % N x F Where:-

F = Conversion factor (6.25)

Percentage (%) Nitrogen = VS - VB x Nacid x 0.028 x 100 W

Where:-

VS = Volume of acid required to titrate sample in milliliters VB = Volume of acid required to titrate blank in milliliters Nacid = normality of acid (0.1N)

W = weight of sample in grams

Therefore, Percentage (%) protein = % Nitrogen x 6.25.

3.7.5 Fat Content Determination

59 The method of Pearson (1973) was employed. The method is based on the principle that non-polar components of the samples are easily extracted into organic solvents. Three (3) grams, (moisture – free) of each sample, was placed into labeled fat-free thimbles. These were then weighed, plugged with glass wool and introduced into the soxlet extractors containing 160 ml petroleum ether (b.p 75^oC). Clean dry receiver flasks were also weighed and fitted to the extractors. The extraction units were then assembled, and cold water was allowed to circulate, while the temperature of the water bath was maintained at 60^oC. Extraction was carried out for eight hours. At the end of this time, the thimbles containing the samples were removed and placed in an oven at 70^oC for three hours and dried to constant weight.

Percentage (%) Fat = Weight of fat x 100 Weight of sample 1

3.7.6 Carbohydrate Content Determination

Carbohydrate content was determined by difference method:

Percentage (%) Carbohydrate = 100 – (% Moisture + % Ash + % Crude fibre + % Crude protein + % Fat).

3.7.7 Determination of Minerals

With the exception of calcium, magnesium and iron, all other mineral contents of these samples were done following the dry ash extraction method outlined by James (1995) and Kirk and Sawyer (1998). A measured weight of these samples were burnt to ashes (as in ash determination) thereby remaining all

60 the organic materials leaving the organic ash. The resulting ashes were each dissolved in 5 mls of dilute (0.1 M) hydrochloric acid solution and then diluted to 100 mls in a volumetric flask. This extract was used in specific analysis for the different mineral elements.