10 - IJA-Vol.3-No.16页
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International Journal of Aquaculture, 2013, Vol.3, No.16, 85
-
91
90
compared with standard.
Formula
Absorption of standard = A nm,
Absorption of sample = Y nm,
“
A” absorption is due to = 1 mL (0.01mg of standard
glucose in 1mL (glucose 10%) (1mg glucose /10 mL)
“1”
is = 0.01 /A,
“
Y” absorption is due to = (0.01/A) ×Y = “F” mg of
amylase in sample
Activity of amylase (F×1000/180(MW of glucose) ×15=
“
Z” IU/ml
3.3.2
Lipases
Ten mmol NaOH was taken in burette for titration and
then 3.5 mL phosphate buffer and 1 ml sample was
taken in glass flask. Then 0.5 mL olive oil was added
in the solution of phosphate buffer and stirred for 30
minutes at 37
℃
in water bath. Then 1 mL acetic acid
was added to this mixture and followed by addition of
3-4
drops of phenolphthalein indicator in enzyme
mixture. The mixture was titrated with 10 mmol
NaOH till the color turns pink.
Formula
Units/ml of enzyme =
= “Y” µM of oleic acid released min
-1
Now Enzyme activity =
= “Z” (IU) /min
3.3.3
Proteases
Total protease activity was evaluated using 1% azocasein
in 50 mmol tris HCl, pH 7.5 (Garcia-Carreno, 1992).
Ten µL of enzyme extract was mixed with 0.5 mL of
buffer (50 mmol Tris-HCl, pH 7.5); 0.5 mL of
substrate solution was incubated for 10 min at room
temperature. The reaction was stopped by adding
0.5
mL 20% trichloroacetic acid and then centrifuged
at 14000×g for 5 minutes. The absorbance of the
supernatant was recorded at 366 nm. Standard curve
was prepared using azocasein as substrate. Values of
protease activity were then calculated by comparing
sample values with those of observed in standard
curve.
3.4
Physico-chemical Parameters
Temperature and dissolved oxygen were measured on
daily basis by using D.O. meter (YSI 55 Incorporated,
Yellow Springs, Ohio, 4387, USA). Electrical
conductivity, total dissolved solids and salinity were
recorded by salinity meter (Condi 330i WTW 82362
Weilheim Germany) while pH was measured by pH
meter ((LT-Lutron pH-207 Taiwan) on weekly basis.
3.5
Statistical Analysis
Data were compared by the analysis of variance
(
ANOVA) at the 0.01 significance level. Significant
differences in mean weights were revealed by Tukey’s
Multiple Range Test for multi-group comparisons. The
data with few replications for the enzyme assays were
analyzed by applying the Least Significant Difference
(
LSD) test, which is less conservative than Tukey’s
test. All data were expressed as Mean ± SD. The SPSS
(
Statistical Package for the Social Sciences) version
16.0
was used for all statistical analyses.
References
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and low temperature on diet acceptability, growth and survival of
mrigal,
Cirrhinus mrigala
,
fingerlings, Pakistan J. Zool., 40(2): 83-90
Bernfeld P., 1955, Enzymes of carbohydrate metabolism, In: Methods in
Enzymology (Colowick, S.P. & Kaplan, N.O. eds), Academic Press,
New York, Vol. 1, pp. 149-541,
(55)01021-5
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intestine of carnivorous fish: structure and functions and the relations
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Open Fish Science Journal, 2: 74-86,
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1874401
X0090201007
Chan A. S., Horn H. A., Dickson K. A., and Gawlicka A., 2004, Digestive
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/
1303-2712-
v12_2_1
used
sample
enzyme
of Vol.
40
× NaOH of
Normality
×
NaOH of Vol.
min 30×
acid)
oleic
254(
M.W.
1000
× Y
