ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA ...

B1.2.3.1.1
The supernatant (designated as the erode extract) was subjected to 80 per cent CNThhS04
saturation and left overnight at 4° C. Protein pellets obtained after centrifugation (12 000
g x 20 minutes) were redissolved in a minimum volume of phosphate buffer (0.02 M,
pH6.9, containing 0.3 M NaCI), dialysed extensively against the buffer (designated the
ammonium sulphate extract) and analysed for amylase inhibitor (AI) activity.
Ion-exchange chromatography
The ammonium sulphate extract was further pu,ified through ion-exchaoge
chromatography (6 cm x I.I cm DEAE-Sephacel, equilibrated with 0.02 M phosphate
buffer). The column was eluted with a linear NaCl gradient of0-0.5 M at the flow rate of
20 mIIh and 5 mI fractions were collected). The absorbance ofthe effluent was monitored
at 280 nm. Individual peaks were pooled and analysed for protein and AI activity.
B1.2.3.1.2 Gel chromatography
Two peaks (A and B, Figure B1-3.2) with AI actlVlty were then separately
chromatographed on a Sephadex G-100 column (35 cm x 1.1 cm, equilibrated with the
phosphate buffer and eluted with the same buffer at a flow rate of 15 mllhr. Five mI
fractions were collected and the absorbance at 280 run was determined). Pooled fractions
were analysed for protein and AI activity. Fractions (A-1 and B-2, Figure B-4) with AI
activities were collected, dialysed extensively, freeze-dried and dissolved in de-ionized
water.
B1.2.3.2 Enzymefmhibitor assay
The method described by Bernfeld (1955) was used to assay for a-amylase and AI
activities. One unit ofamylase is defined as the amount ofenzyme that will liberate 1 !lmol
of maltose from starch under the assay conditions (pH 6.9, 37" C, 5 min). Inhibitory
activity is expressed as the percentage of inhIbited enzyme activity out ofthe total enzyme
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