Method 79 Determination of pepsincellulase digestibility of plant material

See the discussion in Chapter 4, 'Cellulase digestibility'. This method is based on that of Jones and Hayward (1973 and 1975).

Reagents.

• Cellulase solution - dissolve 6.25 g cellulase from Trichoderma viride (Merck Ltd) in 1 l citrate-phosphate buffer, pH 4.6, immediately before use.

• Citrate-phosphate buffer, pH 4.6 - mix together 532.5 ml of 0.1 M citric acid and 467.5 ml of 0.2 M Na2HPO4.

• Citric acid solution, 0.1 M - dissolve 19.212 g citric acid (C6H8O7) in water, and make up to 1 l.

• Disodium hydrogen phosphate solution, 0.2 M - dissolve 28.392 g disodium hydrogen phosphate (Na2HPO4) in water and make up to 1 l.

• Pepsin solution - dissolve 2.0 g pepsin 1:10,000 in 1 l of 0.1 M HCl.

Procedure. Weigh 0.200 g of the dried and ground (to 0.75-1 mm) sample into a screw cap McCartney bottle, graduated at 20 ml. Dispense 20 ml pepsin solution, screw on the cap and shake to mix. Incubate at 40°C for 24 h and shake morning and evening. This is facilitated by holding batches of bottles in a galvanized wire-mesh box (made in-house), and simply inverting a few times. Suck out the pepsin using a sintered glass filter stick, porosity 2 (P100). Rinse the stick with a jet of cellulase solution from a wash bottle, and make up to the 20 ml mark and mix. Incubate for a further 48 h at 40°C, shaking by inversion twice daily. Filter the indigestible residue through a 30-ml sintered glass filter crucible, porosity No. 1 (P160), previously oven-dried and weighed. Wash well with water, then with acetone and leave to air-dry. When no smell of acetone can be detected, dry overnight in an oven at 105°C, then cool in a desiccator and weigh.

Calculation. Subtract the weight of the empty crucible from the weight of the crucible plus residue to obtain the weight of undigested sample W. The amount solubilized is 0.2 - W, therefore the percentage dry matter solubi-lized (x) = [(0.2 - W>/0.2] x 100. This may be correlated with the in vivo dry matter digestibility (y) of grasses using the relationship: y = 0.54x + 35.0.

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