ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA ...

410 DID and the colour remained stable for several hours. Solutions oftrypsin and BAPNA
were prepared as descnbed by Kakade et at (1974). This BAPNA is an artificial substrate
that becomes yellow when it reacts with the trypsin, thus revealing the non-inlubited trypsin
(Kakade et at., 1974).
B.A.2.1.2 Calculation
The change in absorbance (At), owing to trypsin inlubition ml-! diluted sample extract is (AJ,
- Aa) - (A.! - A,), where the subscripts refer to tubes (a) - (d) referred to above. The
percentage inhibition in each sample tube is given by 100 At/(AJ, - Aa). Ifthis is less than 40
per cent or more than 60 per cent, the assay must be repeated to provide a mOTe suitable
dilution (D) ofthe sample suspension. The trypsin inlubitor activity (TIA) was calculated in
tenus ofmgpure trypsin g'! sample as weighed (mg g'!):
2.632·D ·At
TIA = S mg pure trypsin inhibited g'! sample
B.A.2.2 Amylase inhibitor [Bernfeld (1955))
50 g ofdried Amadumbe powder was homogenized and defatted with hexane. The samples
were extracted with distilled water containing 1 per cent polyvinylpolypyrrolidone (pVP).
The resulting crude extract was centrifuged at 10 000 x g for 20 minutes. The precipitate
was discarded and supematant was utilized for enzyme and inhibitory-enzyme assay.
Enzyme and inhibitors, buffered with phosphate buffer, pH 6.9 containing 7 mM NaCI,
were pre-incubated for 10 minutes at 37 0 C. Two per cent starch was utilized as substrate.
After the addition of 10 ml of dinitrosalicylic acid (DNS), reaction was stopped at 100 0 C
and absorbance was measured at 530 DID. One a-amylase unit (lill) was defined as the
amount of enzyme that would liberate 1 J.UIlol of maltose from the starch under the assay
conditions (10 minutes, 37 DC, pH 6.9). The amylase inlubitor's activity (AlA) was
calculated from the equation, through maltose generated:
AlA =
A 530 mnamylase - A 530 run plant extract
A 530 mnamylase
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