References:

- Association of Official Analytical Chemists (AOAC). Official Methods of Analysis,

14th Ed.; Washington DC, 1984; 486
- David Pearson (1976) chemical analysis of foods ISBN 00443014116

Instrument and materials used

- Muffle furnace (Carbolite Aston Lane Hope Sheffield, S30 2RR, England)
- Vacuum Oven (Thermostat vacuum oven Townson & Mercer Ltd. England)
- Weighing machine (Sartorius AG Germany)
- Water bath
- Kjeldahl digestion and distillation apparatus (Labconco)
- LaMotte UV/VIS smart spectrophotometer model 2000

Reagents used
- Concentrated sulphoric Acid-Nitrogen free.
- 50% solution of NaOH containing 5% sodium thiosulphate.
- 2% Boric acid solution.
- 0.1N sulphoric acid.
- Screened methyl red indicator.
- 1g of sodium sulphate and 0.1g of copper II sulphate (Catalyst).

Fat content (%)

10ml of each sample was weighed into a separating funnel, 1ml of 0.88 ammonia was
added and mixed. Exactly 10ml of alcohol (95%) was added and mixed again. Some
25ml peroxide-free diethyl ether was added stoppered and shaken vigorously for 1
minute. Another 25ml of light petroleum (b.p 40-60ºC) was added and shaken vigorously
for 30s. After separation was completed (standing for at least 30 min.), the fat solution
transferred into a suitable flask (previously dried at 100 ºC, cooled and weighed). To the
tube two successive lots of 5ml of mixed ethers were added and transferred (without
shaking) to the flask. 5ml ethanol was added mixed, and then the extraction was repeated
(with 15ml of ether and 15ml of light petroleum) and the subsequent operations. The
extraction was repeated with the ethers once more without addition of ethanol. The
solvents were distilled from the flask, the fat was dried for 1 h at 100 ºC, cooled and
weighed. The flask was washed with light petroleum (should there be any non-fatty
matter), dried, reweighed and the result corrected accordingly.
Protein (%)
The macro Kjeldahl method:

Principle:

This method will not include nitrogen from nitrites and nitrates but will include nitrogen
from proteins, alkaloids, nucleic acids, etc. The organic matter is oxidized by
 concentrated- sulphoric acid in the presence of catalyst and the nitrogen converted to
ammonium sulphate. This is then made alkaline and the liberated ammonia is distilled
and estimated. As a very large part of the nitrogen present in foods is derived from
proteins, the crude protein is estimated by multiplying the percentage of nitrogen by an
appropriate factor.

PROCEDURE:

One (1ml) of the sample was put into a Kjeldahl flask.1g of sodium sulphate and 0.1g of
copper sulphate (catalyst) was added.

Using a measuring cylinder 25ml concentrated sulphoric acid was added to the flask. The
flask was heated gently. The flask was placed in an inclined position. It was swirled
occasionally. When the initial vigorous reaction has died down, the heat was increased
and digestion was continued until the liquid was clear and free from black or brown
Colour. The flask was swirled from time to time to wash down charred particles from the
sides of the flask.

The flask was allowed to cool and the contents was diluted with about 200ml distilled
water and 85ml of 50% NaOH to make the liquid distinctly alkaline. A distillation
apparatus consisting of the flask (500ml capacity), stopper carrying a dropping funnel
and a splash head adaptor was connected to a vertical condenser to which is attached a
straight delivery tube.50ml of 2% boric acid solution was weighed into a 500ml conical
flask, a few drops of screened methyl red indicator was added to it and was placed on the
receiver so that the end of the delivery tube dips just below the level of the boric acid.

The solution was vigorously boiled until about 250ml have distilled over. The receiver
with delivery tube was later removed. The dropping funnel tap was removed and the
source of heat stopped.

The distillate was titrated with a standard acid to pink colour. And the titer value taken.

CALCULATION:

Titer value ×0.0014

%N = × 100
volume of sample taken(1 ml)

Where W is the weight of the sample taken.

% Protein = N × F. Where F is 6.25

Total solid (%)

An empty crucible was weighed (W1), 15ml of the sample was added and evaporated
over a boiling water bath until dryness. This was further dried in an oven at 105 ºC and
weighed again (W2)
 W 2−W 1
Total solid (% )= ×100
Volume of sample taken(15 ml)

Ash (%)
After taking the total solid, the crucibles with the contents were placed into furnace and
heated at 550 ºC for 3 h. after ashing the crucibles were removed from the furnace,
cooled and weighed (W3). W2 is the weight of empty crucible with sample before drying.
W 3−W 1
Ash ( % ) = ×100
W 2−W 1

Carbohydrate (%)
100−(%Moisture+%Ash+ %Protein+%Fat +%Fiber)
Water (%)
100 – Total solid = Water content.

Energy (Kcal)

Energy ( Kcal )=( %Protein ×4 ) + ( %Fat × 9 ) +(%Carbohydrate × 4)

DETERMINATION OF VITAMINS

SAMPLE EXTRACTION

One gram (1 g) of the raw sample was extracted in 10 ml of absolute ethanol by shaking the
sample ultrasonically for 30 minutes. The sample was removed and spun using ultra-modern
centrifuge at 2500 rpm/30 minute. The supernatant was collected and stored at 4 oC for analysis.

STANDARD PREPARATION

A working standard of Vitamins E and K were prepared as follows

An equivalent of 0.75 mg/ml and 0.04 mg/ml was prepared in ethanol for the fat-soluble
vitamins. The standards were shaken for 5 minutes and stored at 4oC.

SPECTROPHOTOMETRIC DETERMINATION OF VITAMINS IN SAMPLE

The observance of the samples and the standards were determined using spectrophotometer. The
instrument was put on and allowed to stabilize for about 15 minutes. The system was blanked
with
ethanol and 0.01 M HCl for vitamins E&K. The absorbance of the standards and the samples
were scanned at 324 nm and 244 nm for vitamins E and K respectively.
Determination of minerals

Concentration of the ions present in the sample was determined by reading their absorbance
using (LaMotte UV/VIS smart spectrophotometer model 2000) and comparing it on the
respective standards. The minerals were determined by absorption/concentration mode and the
instrument readout was recorded for each solution manually. The same analytical procedure was
employed for the determination of minerals in the digested blank solutions and for the spiked
samples.

Proximate composition of Kunun Gyada (g/100g)

S/N Parameter A B
1 Total Solid 25.70 28.15
2 Water 74.30 71.85
3 Ash 1.42 0.46
4 Protein 2.85 2.21
5 Fat 2.88 1.97
6 Fiber 0.80 1.26
7 Carbohydrate 17.75 1.64
8 Energy (Kcal) 108.32 33.13

Mineral composition of Kunun Gyada (mg/100ml)

S/N Parameter A B
1 Magnesium 36.77 31.23
2 Potassium 154.61 141.20

Vitamin composition of Kunun Gyada (mg/100ml)

S/N Parameter A B
1 K 0.06 0.09
2 E 1.02 0.61