BS EN ISO 6647-1:2020 pdf – Rice – Determination of amylose content Part 1: Spectrophotometric method with a defatting procedure by methanol and with calibration solutions of potato amylose and waxy rice amylopectin.
4 Principle Rice is ground to a very fine flour to break up the endosperm structure in order to aid complete dispersion and gelatinization; the flour is then defatted. A test portion is dispersed in a sodium hydroxide solution. An aliquot portion is taken to which an iodine solution is added. The absorbance, at 720 nm, of the colour complex formed is then determined using a spectrophotometer. The amylose mass fraction of the sample is then read from a calibration graph, which is prepared using mixtures of potato amylose and amylopectin to make allowance for the effect of amylopectin on the colour of the amylose–iodine complex of the test solution. 5 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. 5.1 Methanol, a volume fraction of 85 %. 5.2 Ethanol, a volume fraction of 95 %. 5.3 Sodium hydroxide, 1 mol/l solution. 5.4 Sodium hydroxide, 0,09 mol/l solution. 5.5 Detergent solution. Dissolve sodium dodecylbenzene sulfonate corresponding to a concentration of 20 g/l. Just before use, add sodium sulfite to a final concentration of 2 g/l. 5.6 Sodium hydroxide, for protein removal, 3 g/l solution. 5.7 Acetic acid, 1 mol/l solution. 5.8 Iodine solution. Weigh, to the nearest 5 mg, 2,000 g of potassium iodide in a weighing bottle fitted with a stopper. Add sufficient water to form a saturated solution. Add 0,200 g of iodine, weighed to the nearest 1 mg. When all the iodine has dissolved, transfer the solution quantitatively to a 100 ml volumetric flask (6.6), make up to volume with water and mix. Prepare a fresh solution on each day of use and protect it from light. 5.9 Stock potato amylose suspension, free of amylopectin, 1 g/l.
5.9.3 Weigh (6.9) 100 mg ± 0,5 mg of the defatted and conditioned potato amylose into a 100 ml conical flask (6.8). Carefully add 1 ml of ethanol (5.2), rinsing down any potato amylose adhering to the walls of the flask. Add 9,0 ml of 1 mol/l sodium hydroxide solution (5.3) and mix. Then heat the mixture on a boiling water bath (6.7) for 10 min to disperse the potato amylose. Allow to cool to room temperature and transfer into a 100 ml volumetric flask (6.6). Make up to volume with water and mix vigorously. 1 ml of this stock suspension contains 1 mg of potato amylose. If the test samples, the amylose and the amylopectin are conditioned in the same environment, no correction for moisture content is necessary and the results are obtained on a dry milled rice basis. If the test samples and the stocks are not prepared under the same conditions, the moisture content of both the samples and the stocks shall be determined as specified in ISO 712 and the results should be corrected accordingly. 5.10 Stock amylopectin suspension, 1 g/l. Prepare the stock from milled glutinous (waxy) rice with a starch content known to consist of at least 99 % by mass of amylopectin. Steep the milled glutinous rice and blend in a suitable laboratory blender (6.1) to a finely divided state. Remove protein by exhaustive extraction with a detergent solution (5.5) or, alternatively, with a sodium hydroxide solution (5.6. Wash and then defat by refluxing with methanol (5.1) as described in 5.9.1. Spread the deproteinated and defatted amylopectin on a tray and leave for two days to allow evaporation of residual methanol and for moisture content equilibrium to be reached. Carry out the procedure given in 5.9.3, but with amylopectin instead of amylose. 1 ml of this stock suspension contains 1 mg of amylopectin. The iodine binding capacity of amylopectin should be less than 0,2 % (see Annex A). 6 Apparatus Usual laboratory apparatus and, in particular, the following.
6.3 Sieve, size 150 µm to 180 µm (100 mesh to 80 mesh). 6.4 Spectrophotometer, with matching cells, usually of path length 1 cm, capable of measuring absorbance at 720 nm. 6.5 Extraction apparatus, capable of refluxing samples with methanol at a rate of 5 to 6 droplets per second. 6.6? Volumetric? flasks, 100 ml. 6.7 Boiling water bath. 6.8? Conical? flasks, 100 ml. 6.9 Analytical balance, capable of weighing to the nearest 0,000 1 g.