Genomics and Applied Biology, 2017, Vol.8, No.3, 17-25
23
3.3 Inoculation of milled cassava peels samples
Controlled fermentation was carried out using the method described by Okoli et al. (1992) with slight
modification. Twenty grams of the milled cassava peels was weighed rapidly into three fermentation flasks of
volume 250 ml that have been previously sterilized. The flasks containing the mesh were then inoculated with
standardized cells suspension of
Lactobacillus plantarum
containing approximately 5.0 x 10
5
cells/ml without
dewatering the milled samples. The same procedure was repeated for the other isolates:
Geotricum candidum,
Saccharomyces exiguus
and
Corynebacterial manihot.
A mixture of the various all the isolates containing
approximately 5.0 x 10
5
cells/ml was also used as inoculum to ferment the mash, in three different fermenting
flasks. Three fermenting flasks was used for an inoculum per day, there for; a total of 45 fermenting flask was
used for each organisms / treatment for the whole period of fermentation. The control experiment was also set up,
mesh sample of mass twenty grams in forty-five flasks without any inoculum in them (three of the forty-five set
up to be evaluated and discarded daily). The samples were left in the sterile incubator to ferment at ambient
temperature (30 ±2°C) and analysis for linamarin, free cyanide, titratable acidity and pH were taken daily while
the proximate analysis were taken before and after the 96 hours of fermentation.
3.4 Preparation of standard KCN solution
This was prepared by dissolving 125 mg KCN in 0.2 M NaOH. This was then diluted 1: 100 using phosphate
buffer (pH 6.0).
3.5 Preparation of blanks
Blanks used for the colorimetric procedure were prepared as follows. For the total cyanogens determination, 0.4
ml of phosphate buffer (pH 7.0) was mixed with 2.3 ml of phosphate buffer (pH 6.0), 0.1 ml of extraction medium
and 0.6 ml of 0.2 M NaOH in a test tube and treated according to the colorimetric procedure.
For the glucosidic cyanogens (linamarin), 2.7 ml of phosphate buffer (pH 4.0) was mixed with 0.1 ml extraction
medium, 0.6 ml of 0.2 M NaOH and 0.6 ml of KCN standard solution in a test tube.
For free cyanide 3.3 ml of phosphate buffer (pH 4.0), 0.1 ml extraction medium and 0.6 ml KCN standard solution
were mixed together.
Cyanide content (mg HCNequiv/Kg) on dry weight basis was calculated using the expression below.
V = Volume of extraction medium
V' = Volume of adjusted to include sample moisture (ml)
A
590
= Mean absorbance
A
equiv
= Absorbance corresponding to 1 mgHCN
M = Moisture content (%); DW = Dry weight (g); FW = Fresh weight (g); CN = Cyanide content
3.6 Determination of cyanogens
The cyanogens were determined using modified O’Brien et al
.
(1991) method. Five grams of sample was
homogenized in 20 ml of extraction medium (which {extracting medium} was previously prepared by adding 6.75
ml of H
3
PO4 to 200 ml of distilled water) for 15 sec. at low speed, then 2 min. at high speed with 1 min. rest in
between periods. The sample was subsequently centrifuged for 10min. at 4000 rpm. The supernatant was collected
for analysis for cyanogens (glucoside cyanogens) and residual cyanide (HCN).
