CHAPTER THREE

MATERIALS AND METHOD

3.1 Apparatus
 Weighing balance(Gallenkamp)
 Electric coil heater (Gallenkamp)
 Volumertic flask 250ml(pyrex)
 Markham distillation apparatus (pyrex)
 Incubator(DNP-9052)
 Filter paper (whatman)
 Glass rod
 Spatula
 Fume cupboard
 pH meter(coring,philon meter 458)
 Conical flask 100ml,250m (pyrex)
 Autoclave (webeco, Germany)
 Measuring cylinder 10ml, 100ml, 500ml,1000ml(pyrex)
 Beaker 200ml,500ml (pyrex)
 Masking tape
 500ml Kjedhal flask (Tecator)
 Aluminum foil
 Cotton wool
 Pestle and mortar
 Micropipette and tips(Fisher brand)
 Dessicator
 Hot air oven(Driver box DHC9053)
3.2 Reagents
 Potassium sodium tartrate(BHD)
 Arsenomolybdic acid (May and Bakers)
 Phenol sulphuric acid (May and Bakers)
 Copper tartrate (May and Baker)
 Potassium hydroxide (May and Bakers)
  Boric acid (BDH)
 Sodium hydroxide (May and Baker)
 Hydrochloric acid(May and Baker)
 Concentrated sulphuric acid (May and Baker)
 Ethanol (May and Baker)
3.3 Preparation of Reagents
3.3.1 Preparation of 12.5% NaOH
12.5% of NaOH will in 80%of distilled water and the volume was make up to 100ml with
distilled water.
3.3.2 Boric Acid
2g of 2% stock of Boric acid was weighed and dissolve in some little amount of distilled water
and the volume was made up to 100ml with distilled water.
3.3.3 Mixed indicator
Methyl red was weighed to 0.01gand dissolve in 100ml of ethanol. Weigh
0.01bromocresol(BCG) and dissolve separately in 100mlof ethanol. The two solutions were
mixed together.1ml of ethanol mixture was added to the 2% stock solution of Boric acid.
3.3.4 Fehling's solution (A&B)
Fehling's solution A
3.4g of copper sulphate pentahydrate was weighed and dissolve in 500ml of distilled water.
Fehling's solution B
17.3g of potassium sodium tartrate and 50g of sodium hydroxide is dissolved in 500ml of
distilled water
3.3.5 Preparation of inoculum
Baker's yeast (Saccharomyces cereviseae) was obtained as a stock culture from the department
of Microbiology, Modibbo Adama University of Technology.
3.4 Collection and Preparation Of Sample
Sugarcane pith was collected from Savannah sugar company, Numan while groundnut shell was
collected from Hong local government area of Adamawa state from a local farm. Baker's yeast
(Saccharomyces cereviseae) was collected from the department of Microbiology, Modibbo
Adama University of Technology, Yola. Both the Bambaranut shells and the sugarcane pith were
grounded separately using mortar and pestle. The ground powder of both sugarcane pith and
Bambaranut was sundry.
3.5 Pretreatment of Sample
30g of powdered sugarcane pith and Bambaranut were combined together in 50:50 ratio, and
poured into Five labelled (0,2nd,4th,6th,8th day) fermenting cornical flaskof 500ml each. 150ml
of 12.5% of NaOH was added to each of the flask to 4.6 with 25%HCL. Cover with aluminium
foil and autoclave at 15psi at 121 degree centigrade for 15min (Raghavendra et al.,2007).
30g of combined sugarcane pith and bambaranut powdered form were weighed and pour into
five labelled (0,2nd,4th,6th,8th day) fermenting cornical flask of 500ml each. To each of the
labelled cornical flask,150ml of 12.5% of NaOH was added. The pH of each of the cornical flask
was adjusted to 4.6 with 25%HCL and cover with aluminium foil and autoclave at 15psi at
121degree centigrade for 15min (Raghavendra et al., 2007).
3.5.1 Fermentation process
4ml of inoculum from stock of Saccharomyces cereviseae was added to each of the labelled flask
containing the pre-treated sample and incubate at 30degree centigrade of a period of 0, 2,4,6,
and 8 days of fermentation respectively. At the end of the fermentation period of each of the
flasks, the biomass was placed on a filter paper and wash with distilled water at room
temperature by analysis.
3.5.2 pH Determination
The pH of the fermented sugarcane pith and bambaranut shells was determined immediately
after stopping the fermentation on the 0,2nd,4th,6th and 8th day.
3.5.3 Protein content determination
After the pH is being determined, the sample was dried in an oven at 40 degree centigrade. Using
Kjedhal method, the protein content was determined.
Procedure
4g of the fermented sugarcane pith and bambaranut shells (combined) was weighed and transfer
to 500ml Kjeldahl flask. 10g of sodium sulphate and 0.5g of copper sulphate are added into the
flask and mix with 25ml of concentrated sulphuric acid. the flask was slanted and heated for 3hrs
(digestion), there was change in color to dark brown, the solution was allowed to cool and then
transfer into 250ml volumertic flask. A Markham's distillation apparatus was set up with the tip
dipped into a receiving flask containing 5ml of Boric acid indicator passed, pass through the set
up 5ml of the dilluted digest. 5ml of 50% sodium hydroxide (NaOH) was pour through the set-
up. The set up is allow to continue for 2-3min untill there is a color change from colorless to
green and the distillate double it's quality (10m). The distillation was stopped and titrate against
0.01N hydrochloric acid solution (HCL). At the end point an immediate color change was
observe from Green to red marks. Utilize the same procedure was utilized for blank, using starch
instead of fermented sugarcane pith and bambaranut shells in combined form. The titer value
was obtained from the tests sample and the blank was used to calculate the percentage nitrogen
value of test sample the value was multiplied with the value of blank by 6.25 in order to obtain
the crude protein content.
Formula: % Nitrogen Value=1.4N (V1-V2) ⁄W
Where:
W=weight of the sample
N= normality of sodium hydroxide
V²= titre value of Hydrochloric acid (HCL) obtained from test sample
V²= titre value of Hydrochloric acid (HCL) obtained from blank samples
3.6 Moisture content determination
An empty aluminium dish was weighed and recorded, 4g of fermented sample was poured into
the aluminium dish. It was then being placed in an oven and dry at 105C for 3hrs, after which it
was removed and weighed.
Moisture content% (MC)=(W1-W2)X 100
W1-W
Where:
W=weight in gram of aluminium dish
W¹= weight in gram of aluminium dish+ sample before drying
W²= weight in gram of aluminium dish+dried sample
3.7 Total Sugar (Reducing and non-reducing sugar)
The total sugar was determined using the spectrophotometric method of sugar analysis by Nelson
N.A (2010).
3.7.1 Total Sugar
To measure the total sugar in the sample, phenol sulphuric acid was used. Samples for the
various day of fermentation were taken into the testube from 0.2ml to 1ml, up to 10ml volume
was made with distilled water added. Then 1ml of 5% phenol and 5ml of concentrated H2SO4
were added. All the testube where stand for 10-15min. Then the absorbance of each sample and
standard (glucose) were measured at 550nm.
3.7.2 Reducing Sugar
From the various days of fermentation (0,2,4,8) 100ml of the samples were taken into the
testubes and the sugars were extracted with hot 80% ethanol twice (5ml each time). The
supernatant was collected and evaporated, then keep on a water bath at 80C. 10ml of distilled
water use to dissolved the sugars. Pipette 0.2ml, 0.4ml, 0.6ml, 0.8ml and 1ml of working
standard solution into a series of the testube. Make the volume in both standard and sample tubes
up to 2ml with distilled water. 2ml of distilled water was pipette into a separate tube to set a
blank. Add 1ml of alkaline copper tartrate reagent in each tube. The tube should be placed in a
boiling water bath for 10min. Then the tube was cooled added 1ml of arsenomolybdic acid
reagent was added into each tubes. The absorbance of the standard and test sample at 660nm was
measured.
3.7.3 Non-Reducing Sugar
The non-reducing sugar of this experiment was determined by subtracting the reducing sugar
from the total sugars. Non-reducing sugar (Total sugar-Reducing sugars).