International Journal of Aquaculture 2015, Vol.5, No. 41, 1-20
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tube remained well stoppered. After cooling in ice
water for 15 minutes, the tube was allowed to adjust to
room temperature. Once the tube reached room
temperature, the content of the tube was mixed and
centrifuged for 30 minutes at 3000g. 1 ml of the
supernatant was transferred to another test tube and
1.5ml of 2, 2’- Bipyridine solution added. The
absorbance was measured at 519nm against distilled
water.
2.4.2 Tannin
Tannin in raw, processed and experimental diets was
determined using methods described by AOAC (2006).
About 2g of feed was defatted for 2 hours using
Soxhlet extraction apparatus. The residue was dried in
oven for 12 hours at 100°C, boiled with 300ml of
distilled water, diluted to 500ml in standard
volumetric flask and filtered through non -absorbent
cotton wool. A volume of 25ml of the infusion was
measured into 2 liter porcelain dish and titrated with
0.1N potassium permanganate (0.1N potassium
permanganate was standardized against 0.lN oxalic
acid) until the blue solution turned green; then few
drops of 0.lN potassium permanganate was added.
The difference between the two titrations was
multiplied by 0.006235 to obtain the amount of tannin
in the sample using equation (1).
0.lNoxalic acid=0.006235g tannin…………………….(1)
2.4.3 Total oxalate
The total oxalate of the powdered samples was
determined by the modified method of Abeza et al.
(1968). About 2g aliquot of the sample was weighted
into a 250 ml flash; 190 ml distilled water and 10ml of
6M hydrochloric acid were added. The mixture was
digested for 1 hour on boiling water bath, cooled and
transferred into a 250 ml volumetric flash, diluted to
volume and filtered. Four drops of methyl red
indicator were added followed by concentrated
ammonia until the solution turned faint yellow. It was
then heated to 100°C, allowed to cool and filtered to
remove precipitate containing ferric ions. The filtrate
was boiled, 10 ml of 5% calcium chloride added with
constant stirring and was allowed to stand overnight.
The mixture was filtered through whatman No. 40
filter paper. The precipitate was washed several times
with distilled water. The precipitate was transferred
quantitatively to a beaker and 5ml of 25% sulphuric
acid was added to dissolve the precipitate. The
resultant solution was maintained at 80°C and titrated
against 0.5% potassium permanganate until the pink
colour persisted for approximately one minute. A
blank was also run for the test sample. From the
amount of potassium permanganate used, the oxalate
content of the unknown sample was calculated using
equation.
1ml of KMn04=2.2mg oxalate…………………… (2)
2.5 Determination of phytase activity
Phytase activity in feed samples were determined
modified by the Relative Method of BASF (1997)
with slight modification to methods by Engelen et al.
(1994). About 100 g feed was milled to a particle size
less than 0.5 mm. Two 5.0 g portions of each sample
of feed was weighed with an accuracy of 10 mg into
an Erlenmeyer flask. 50.00 ml acetate buffer was
metered by a dispenser into each sample, and the
mixture was then stirred on a magnetic stirrer for 60
min. The stirring was followed by decantation into 10
ml centrifuge tubes and centrifugation at 4000 rpm
(equivalent to about 2500g) for 20 minutes. The
centrifugate was then diluted with buffer using the
dilutor to a content of about 0.02 FTU/ml. 2.00 ml of
each of the two solutions was pipetted as sample and
sample blank into a 10 ml centrifuge tube. For the
blank, 2.00 ml portions of acetate buffer were pipetted
into two 10 ml centrifuge tubes. One centrifuge tube
was incubated, and the other centrifuge tube was
treated in analogy to the blanks as enzyme standard.
The centrifuge tubes with the enzyme, sample blank
and control solutions were each placed at a defined
time interval (e.g. every 10 sec) in a water bath at 37.0
+/- 0.1°C and equilibrated for exactly 5 min. Then, at
the same time intervals (every 10 sec), 4.00 ml sodium
phytate solution (equilibrated at 37.0 +/- 0.1°C) was
added by a dispenser and mixed. After incubation for
exactly 60 min, the reaction was stopped, again at the
same time intervals (every 10 sec), with 4.00 ml stop
reagent and mixed to produce a colored complex with
the phosphate formed. After waiting for at least 10
min, the solutions were centrifuged at 4000 rpm
(equivalent to about 2500 g) for 20 minutes and then
the absorbance at a wavelength of 415 nm was
measured in a spectrophotometer against air. The
enzyme phytase liberates inorganic phosphate from
the substrate sodium phytate during incubation and the
intensity of
the yellow color
of
the
