Method 103 Determination of total nonstarch polysaccharides

This is based on the procedure by Englyst and Cummings (1984) who performed a GLC analysis of the alditol acetate derivatives of the constituent sugars. It incorporates slight modifications as currently carried out at IGER, Aberystwyth (Paul Thomas, IGER, Aberystwyth, 2001, personal communication). The starch is first dispersed with dimethyl sulphoxide, which disrupts intermolecular hydrogen bonds, followed by hydrolysis with a-amylase and pullulanase. The former hydrolyses the straight chain a-1-4-glycosidic bonds of amylose, the latter, also known as alphadextrin 6-glucanohydrolase, is a specific enzyme for the hydrolysis of the branching a-1-6-glycosidic bonds in the amylopectin component of starch.

Reagents.

• Acetate buffer - 0.1 M sodium acetate solution adjusted to pH 5.2.

• P-d-Allose internal standard solution, 1 mg ml-1 - dissolve 200 mg P-d-Allose (Sigma Cat. No. A-6390) in 50% (v/v) saturated benzoic acid solution and make up to 200 ml. Store in the dark. (This is sufficient for at least 36 tests - it is expensive, so make just a sufficient amount.)

• Dimethylsulphoxide - (DMSO, Sigma ACS Reagent, Cat. No. D8779).

• Ethyl acetate

• Mixed enzyme solution - either make up a mixed enzyme solution containing 5000 units of a-amylase and 5 units of pullalanase per millilitre acetate buffer (original method), or, as is usual, separately add 0.5 ml a-amylase reagent and 0.1 ml pullulanase reagent per sample.

a-Amylase reagent - (EC 3.2.1.1.), capsules (Pancrex V, 300 capsules, high potency pancreatin. Contains: amylase, 9000 BP units; lipase, 8300 BP units, free protease, 430 BP units. Manufacturer: Paines and Byrne Ltd, West Byfleet, Surrey, UK; Supplied by Boots Chemists). For 32 samples, immediately before use dissolve four capsules in 18 ml water at room temperature and centrifuge.

Pullulanase reagent - (EC 3.2.1.41.), suspension in 3.2 M (NH4)2SO4, pH 6.2, 5 units per mg protein (Sigma Cat. No. P5420). For up to ten samples, make up 0.010 ml pullulanase suspension to 1 ml with acetate buffer, pH 5.2, and keep refrigerated until use. Sulphuric acid, 12 M H2SO4

Equipment.

• Three-place magnetic stirrer-hotplate

Procedure (enzyme and acid hydrolysis). Weigh accurately 50-1000 mg representative sample (freeze dry, ball mill or homogenize if necessary, but do not oven-dry) containing ^150 mg starch and ^50 mg NSP into a 50-60-ml screw-top centrifuge tube (borosilicate glass, approximately 200 x 26 mm) and add an approximately 12 mm PTFE-coated magnetic stirring bar. Samples with 90-100% DM and <2-3% fat may be analysed directly, which is usually the case, otherwise add 40 ml acetone, stir for 30 min, centrifuge and remove as much supernatant as possible, without disturbing the residue, by means of a plastic Pasteur pipette or glass capillary connected via a Buchner flask (to retain the solvent) to a vacuum line. Evaporate off the acetone using a water-bath at approximately 65°C in a fume cupboard. (This may be an arrangement of three 2-l glass beakers of water on a three-place magnetic stir-rer-hotplate, which will allow mixing until dry. A J-cloth may be placed in the bottom of the beaker to prevent breakages, and the beaker should be covered with a watchglass to reduce evaporation when used as a boiling water bath (see below). Occasionally check the water level to ensure it does not boil dry.)

Add 2 ml DMSO to the centrifuge tube, replace the cap and heat for 1 h after returning to the boil in a boiling water bath on a stirrer-hotplate (as described above) with continuous stirring. Without cooling, from a dispenser add 8 ml acetate buffer at 50°C and vortex mix immediately. After cooling to 45°C, immediately add 0.5 ml a-amylase reagent and 0.1 ml pullulanase reagent. Incubate for 16-18 h at 45°C with regular mixing. Remove from the incubator and add 40 ml absolute ethanol, mix thoroughly and allow to stand for 1 h at ambient temperature before centrifuging for approximately 10 min, or until a clear supernatant is obtained. Remove as much of the supernatant as possible by aspiration into a Buchner flask for safe disposal. Wash twice with 50 ml 85% (v/v) ethanol by mixing well, centrifuging, and removing the solvent as above. Add 40 ml acetone to the residue, and stir thoroughly on the magnetic stirrer-hotplate for 5 min. Check that all the stirrer-bars are rotating or oscillating well, and if necessary, adjust the position of the beaker to achieve this. Aspirate the solvent into a separate Buchner flask for safe disposal or redistillation and re-use. Dry in a beaker of water at 65°C in a fume cupboard.

Add 2 ml of 12 M sulphuric acid to the dried residue and disperse using a vortex mixer; inspect the bottom of the tube to check that no unmixed sample remains. Heat at 35°C for 1.25 h to solubilize the cellulose, then quickly dispense 22 ml water and mix. Heat in a boiling water bath for 2-2.25 h timed from re-boiling, with continuous magnetic stirring. Place in water at room temperature to cool, and add 5 ml P-d-allose internal standard solution and mix; this solution may be refrigerated until required for analysis. Take a 1-ml aliquot of the hydrolysate for preparation of alditol acetates, and retain the rest if required for optional determination of interference from uronic acids (see Englyst and Cummings, 1984).

Preparation of alditol acetates Reagents.

• Acetic anhydride

• Ammonia solution, 12 M and 3 M - dilute 92 ml of ammonia solution, 0.910 g cm3, approximately 13 M, to 100 ml with water to give a 12 M solution. Further dilute 25 ml to 100 ml to give a 3 M ammonia solution.

• 1-methylimidazole - (N-methylimidazole, NmetIm; Sigma Cat. No. M8878, 500 ml) Safety note: extremely dangerous to mucous membranes of upper respiratory tract, burns mouth, oesophagus and skin, and destructive to eyes. Use only in a fume cupboard/hood. Flush contaminated skin with copious amounts of water.

• Mixed standard - dissolve 200 mg arabinose, 100 mg galactose, 700 mg glucose, 100 mg mannose, 100 mg rhamnose and 500 mg xylose in 50% saturated benzoic acid solution and make up to 100 ml; keep refrigerated in the dark - it has a long shelf-life.

• Potassium hydroxide solution, 7.5 M - dissolve 210 g potassium hydroxide pellets in water and make up to 500 ml and mix. Safety note: this is highly caustic and PPE must be worn.

• Sodium tetrahydroborate(III) reagent - freshly prepare by dissolving 50 mg sodium tetrahydroborate(III) per ml 3 M ammonia solution (0.1 ml reagent used per sample). Note: original method used 100 mg ml-1 concentration.

Equipment.

• Crimp-top vials - suitable vials for GLC are available in the UK from various suppliers, including Vials Direct, Cat. No. V2.3T, PO Box 117, Macclesfield SK11 8DH.

Procedure. Transfer 1 ml hydrolysate solution to an approximately 100 x 21 mm borosilicate centrifuge tube and add 0.2 ml of 12 M ammonia solution and mix; test a small drop to ensure that it is alkaline. Add 0.1 ml of a freshly prepared solution of sodium tetrahydroborate(III) reagent and 1-5 pl of octan-2-ol to prevent foaming, then vortex well to mix. Heat for 1 h at 40°C. Next add 0.1 ml glacial acetic acid, vortex-mix and remove a drop to check the pH is acid. Transfer 0.5 ml of the acidified solution to a smaller screw-cap glass tube and add 0.5 ml 1-methylimidazole, 5 ml acetic anhydride, vortex-mix and leave at room temperature for 10 min. Add 0.9 ml ethanol, vortex-mix, leave 5 min before adding 5 ml water and then vortex-mix. Place in an ice-water bath for 5 min, then add 5 ml 7.5 M KOH solution, leave 5 min, then add a further 5 ml 7.5 M KOH solution. Cap the tube and invert to mix, then leave the tube to allow the two liquid phases to separate. Transfer the top solvent layer to a small vial, crimp on the top and store at 5°C before injecting about 1-2 pl onto the gas chromatograph.

Take 0.25 ml of the mixed sugars standard solution, add 0.25 ml allose internal standard solution and 0.5 ml 2 M sulphuric acid, mix and substitute instead of the hydrolysate solution in the above procedure for preparation of alditol acetates. The concentration of sugars should give a linear relationship with peak area over the normal ranges. The GLC determination conditions will depend on the instrument, but the following are suggested:

Injector temperature: 260°C

Oven temperature: 220°C

Detector (FID) temperature: 250°C

Carrier gas: helium.

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