Method 74 Determination of neutral cellulase plus gamanase digestibility NCGD of feeding stuffs

The method is based on that of MAFF (1993b) and is discussed in Chapter 4.

It is intended for samples of compound feeds or feed mixtures.

Apparatus.

• Filter tubes - a special sintered borosilicate glass filter tube with Suba-Seal and plastic cap is available from Soham Scientific, Unit 6, Mereside, Soham, Ely, Cambs CB7 5EE, UK, and is shown in Fig. 7.1.

Reagents.

• Enzymes - test kits of consistent quality are obtainable from Biotal Limited, 5 Chiltern Close, Cardiff, CF4 5DL, UK; tel. +44 (0)2920 766716, fax +44 (0)2920 747414. The cost is approximately £327 + VAT per kit for 250 tests.

• Acetate buffer solution, pH 4.8 - dissolve 1.36 g of sodium acetate in 500 ml of distilled water, add 0.6 ml glacial acetic acid and dilute to 1 l. Check the pH now and before use, and adjust to pH 4.8 with sodium hydroxide solution.

• Acetone, commercial 'drum' grade.

• Amylase solution - dissolve 2 g of a-amylase in 90 ml distilled water and filter. Add 10 ml of 2-ethoxyethanol to the filtrate, and store at 5°C. Prepare fresh daily.

95 mm

j

k

34

mm

1

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Sintered glass filter P160/Porosity No. 1 30 mm 0

Suba-seal

Fig. 7.1. Borosilicate tube with sintered glass filter and end seals.

• Buffered cellulase solution - transfer 20 g cellulase (also called polysac-charase in the kit) and 0.1 g chloramphenicol to a 2-l wide-neck conical flask. Add 1 l of acetate buffer solution, shake and incubate for at least 1 h at 40°C.

• Cellulase-gamanase solution - add nine volumes of cellulase solution to one volume of the gamanase preparation and mix thoroughly. Filter through a Whatman GF/A glass micro-fibre filter circle held in a Hartley type three-piece funnel.

• Chloramphenicol, (D(-)-threo-2-dichloroacetamido-1-p-nitrophenylpropane-1,3-diol; C11H12Cl2N2O5, molar mass 323.13). Note: ingestion may cause a toxic effect on bone marrow, and contact with skin may cause an allergic response.

• Neutral detergent solution - dissolve 93 g EDTA disodium salt, and 34 g disodium tetraborate decahydrate (Na2B4O7.10H2O) in water on a stirrer/hotplate. Add 150 g sodium dodecyl sulphate (sodium lauryl sulphate, CH3(CH2)11OSO3Na)) and 50 ml of triethylene glycol (HO(CH2)2O(CH2)2O(CH2)2OH). (Note: the published method specifies the toxic 2-ethoxyethanol (CH3CH2OCH2CH2OH)). Add a solution of 22.8 g of sodium dihydrogen phosphate, anhydrous (NaH2PO4) prepared by dissolving in water on a stirrer/hotplate. Dilute to 5 l and mix thoroughly. Adjust the pH to 6.9-7.1 if necessary. Note: sodium dodecyl sulphate dust irritates the lungs, therefore wear dust masks when weighing and use dust extraction fans.

• Petroleum spirit (light petroleum), boiling range 40-60°C

Procedure. Prepare a filter tube by placing in a cool muffle furnace, increase the temperature to 500°C, and maintain it for 30 min. Remove and allow to cool in a desiccator. To avoid damaging the sinter, do not allow the temperature to reach 515°C. Weigh 0.5 g of the sample, as received, but ground to 1 mm, into the crucible. At the same time, weigh a separate portion for DM and ash determination. Wash the sample in the filter tube with 3 x 25 ml portions of petroleum spirit. Suck as dry as possible under gentle vacuum and complete the removal within 10 min at 60°C in an oven specified for use with flammable solvents.

Carefully brush the fat-free residue into a 150-ml flat-bottom flask, add 25 ml of neutral detergent and swirl to mix. Place in the heating unit and attach the reflux condenser, ensuring a steady flow of cold water. Bring to the boil and reflux for 30 min. Swirl occasionally to prevent overheating the sample and ensure adequate mixing. Then turn off the heat and add 25 ml of cold neutral detergent solution followed by 2 ml of amylase solution. Again heat to boiling and reflux for a further 30 min, occasionally swirling the contents of the flask.

Turn off the heat and immediately filter through the same filter tube previously used for the fat extraction. Wash the residue thoroughly with at least 3 x 20 ml hot distilled water, as it is essential to remove all the neutral detergent solution. Moisten a Suba-Seal and carefully push it into the bottom end of the filter tube as far as the sinter disc. Use dispensers to add 25 ml of distilled water at 80°C followed by 2 ml of amylase solution. The force of the jet from the dispensers should be enough to agitate the contents; if not, perform an additional mixing; allow to stand for 15 min. Remove the Suba-Seal and apply suction to remove the amylase solution. Replace the Suba-Seal and, using a dispenser, add 30 ml buffered cellulase-gamanase solution to the residue. Secure the polythene cap on the top of the tube and shake to mix the fibres thoroughly with the enzyme solution. Incubate at 40°C (± 2°C) for 40 h, shaking morning and evening to ensure adequate mixing.

Remove both the Suba-Seal and polythene cap and place the filter tube in the rubber cone adapter (42 x 27 mm) used with the adapter funnel attached to the Buchner flask, or other suitable device. Wash any particles from the polythene cap into the filter tube, apply just sufficient suction to remove the gamanase-cellulase solution, then wash the residual undigested fibre with hot distilled water (approximately 80°C). Finally wash well with acetone, leave to air dry in a fume cupboard, and when no smell of acetone can be detected, dry in an oven overnight at 100°C (±2°C). Cool in a desiccator and weigh the filter tube plus residue.

Place the crucible and its contents of NCGD fibre in a cool muffle furnace. Increase the temperature of the furnace to 500 ±5°C; keep at this temperature (for approximately 3 h) until ashing is complete. It is important not to overshoot the temperature, because the sintered disc will be damaged at ^515°C. Remove the crucible from the muffle furnace, cool in a desiccator and weigh.

Calculation. Subtract the weight of crucible plus ash from the weight of crucible plus residue to obtain the weight of indigestible organic matter in the 0.5 g sample ('as received'). Divide by the sample weight and multiply by 100 to get the % indigestible organic matter in the 0.5 g sample ('as received'). This must be corrected for moisture in the sample, therefore using the value from a separate moisture determination, multiply by 100/(100-%moisture) to obtain the % indigestible organic matter in sample DM. From the total ash determination, calculate the % total ash, and correcting for sample moisture as above, express the total ash as % in sample DM. The NCGD = 100 - (% indigestible organic matter in DM + % total ash in DM).

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