Moisture, crude protein, ash, crude fat and crude fibre were
estimated using AOAC (1990) method. Carbohydrates were
calculated by difference. The proximate principles ie.
moisture, crude protein, ash, crude fat and crude fibre were
summed up and subtracted from 100. In vitro protein
digestibility was estimated by the method of Akeson and
Stachmann (1964) modified by Singh and Jambunathan
(1981). 0.5 g was digested with 5 ml of Hcl solution (pH2.0)
containing 2 mg of pepsin (P2000 HD821, Sigma Chemicals
Limited, USA). The flask was incubated in a water bath
shaker for 16 h at 37 °C. Two ml of pancreatin (P7547
H0159, Sigma Chemicals Limited, USA) solution was added
and the contents were further incubated for 24 h. (Pancreatin
solution was prepared by dissolving 50 mg of pancreatin in
100 ml of 0.1 ml borate buffer (pH 6.8), containing 0.025 M
CaCl2. After 24 h, the reaction was stopped by adding 7.0 ml
of 10% (W/V) trichloroacetic acid (TCA) and the suspension
was centrifuged (at 10,000 rpm) for 15 min. The residue was
washed twice with 5 ml of 5% TCA. Finally this 25.0 ml of
volume was taken to estimate the nitrogen content by the
standard microkjeldhal method.