ANNEX ISupplementary provisions
ANNEX I Determination of starch content and its degradation products including glucose 1. PURPOSE AND FIELD OF APPLICATION (a) The method permits the determination of the starch content, its degradation products including glucose, hereafter referred to as ‘starch’. (b) ‘Starch’ content referred to in point (a) is equal to value E, as calculated in point 6(a) of the present Annex. 2. PRINCIPLE The sample is broken down by means of sodium hydroxide and the ‘starch’ divided into glucose units with amyloglucosidase. The glucose determination is performed by the enzymatic route. 3. REAGENTS (Use double-distilled water) 3.1. 0,5 N sodium hydroxide solution (0,5 mol/l). 3.2. 96 % (minimum) glacial acetic acid. 3.3. Solution of amyloglucosidase: Immediately before use, dissolve approximately 10 mg of amyloglucosidase (EC 3.2.1.3) (60 U per mg) in one ml of water ( 1 ) . 3.4. Triethanolamine buffer solution: dissolve 14,0 g of triethanolamine hydrochloride [tris(2-hydroxyethyl)ammonium chloride] and 0,25 g of magnesium sulphate MgSO 4 7H 2 O) in 80 ml of water, add approximately 5 ml of 5 N sodium hydroxyde solution (5 mol/l) and adjust the pH to 7,6, using a 1 N sodium hydroxide solution (1 mol/l). Make up to 100 ml with water. This buffer solution can be kept for at least four weeks at 4 °C. 3.5. NADP (nicotinamide adenine dinucleotide phosphate, disodium salt) solution: dissolve 60 mg of NADP in 6 ml of water. This solution can be kept for at least four weeks at 4 °C. 3.6. ATP (adenosine-5′-triphosphate, disodium salt) solution: dissolve 300 mg of ATP. 3H 2 O and 300 mg of sodium hydrogen carbonate (NaHCO 3 ) in 6 ml of water. This solution can be kept for at least four weeks at 4 °C. 3.7. Suspension HK/G6P-DH [Hexokinase (EC 2.7.1.1) and glucose-6-phosphate-dehydrogenase (EC 1.1.1.49)]: suspend 280 U HK and 140 U of G6P-DH in 1 ml of 3,2 M ammonium sulphate solution. This suspension can be kept for at least one year at 4 °C. 4. APPARATUS 4.1. Magnetic stirrer water bath at 60 °C. 4.2. Magnetic rods. 4.3. UV spectrophotometer with 1 cm optical cells. 4.4. Pipettes for enzymatic analysis. 5. METHOD 5.1. The sample is digested in sodium hydroxide and the ‘starch’ subject to enzymatic hydrolysis 5.1.1. Select the weight of sample as follows, according to the presumed ‘starch’ content (the ‘starch’ content must not exceed 0,4 g per sample) as follows: Presumed ‘starch’ content of product in g/100 g Approximate weight of sample in g (p) Volume of graduated flask in ml Dilution factor up to 1 litre (f) > 70 0,35-0,4 500 2 20-70 max. 0,5 500 2 5-20 max. 1 250 4 < 5 max. 2 200 5 5.1.2. Weigh the sample exactly to 0,1 mg. 5.1.3. Add 50 ml of 0,5 N sodium hydroxide solution (point 3.1) and stir continuously for 30 minutes in the water bath (point 4.1) with a magnetic stirrer at 60 °C. 5.1.4. Add a few ml of concentrated acetic acid (point 3.2) and bring the pH to 4,6 to 4,8. 5.1.5. Place in the water bath with the magnetic stirrer (point 4.1) at 60 °C, add 1,0 ml of enzyme solution (point 3.3) and allow to react whilst stirring continuously for 30 minutes. 5.1.6. After cooling, transfer quantitatively to a graduated flask (point 5.1.1) and make up to the mark with water. 5.1.7. If necessary, filter through a fluted filter (see Note 1). 5.2. Quantitative determination of glucose 5.2.1. The test solution must contain 100 to 1 000 mg of glucose per litre, which corresponds to a ΔΕ 340 of between 0,1 and 1,0. The absorbence of the test solution in a 1 + 30 dilution with water must not exceed 0,4 (measured against air), at 340 nm. 5.2.2. Bring the buffer solution (point 3.4) to ambient temperature (20 °C). 5.2.3. The temperature of the reagents and of the sample must be between 20 and 25 °C. 5.2.4. Measure the absorbence at 340 nm against air (i.e. with no optical cell in the reference path). 5.2.5. Proceed in accordance with the pipetting chart below: Pour into the optical cells Control (ml) Test (ml) Buffer (reagent 3.4) 1,00 1,00 NADP (reagent 3.5) 0,10 0,10 ATP (reagent 3.6) 0,10 0,10 Test solution (5.1.6 or 5.1.7) — 0,10 Double-distilled water 2,00 1,90 Mix and, after about three minutes, measure the absorbence of the solutions (E 1 ). Initiate the reaction by adding: HK/G6P.DH (reagent 3.7) 0,02 0,02 Mix, allow the reaction to proceed to completion (approximately 15 minutes) and measure the absorbence of the solutions (E 2 ). If the reaction has not stopped after 15 minutes read absorbences at five-minute intervals until the rate of increase is constant. Then extrapolate backwards to the time of addition of suspension (referred to in point 3.7 (see Note 2). 5.2.6. Calculate the absorbence differences for reagent blank and sample (E 2 – E 1 ). Subtract absorbence difference of the reagent blank (ΔΕ reagent blank) from that of the sample (ΔΕ sample): ΔΕ = ΔΕ sample – ΔΕ reagent blank This difference gives the glucose content of the test solution: Glucose content contained in test solution, g/l Gl = ((3,22 × 180,16)/(6,3 × 1 × 0,1 × 1 000)) × ΔΕ 340 = 0,921 × ΔΕ 340 (3,22: volume of the solution to be measured; 1: cell thickness; 0,1: volume of the sample solution; the molecular weight of glucose is 180,16 (g/mol). 5.2.7. If, for any reason, a measurement cannot be made of 340 nm, the measurement may be made at a wavelength of 365 nm or 334 nm, in which case the figure 6,3 in the formula for Gl above should be replaced by the figure 3,5 or 6,18 respectively. 6. CALCULATION AND EXPRESSION OF RESULTS (a) E = ‘Starch’ content in g/100 g: E = ((100 × 0,9 × Gl)/(p × f)) (b) Z = ‘Glucose’ content in g/100 g: Z = ((100 × Gl)/(p × f)) where: Gl = glucose in G/l (5.2.6); F = dilution factor (5.1.1); p = weight of sample in g; 0,9 = glucose conversion factor for starch. Notes: (1) Where a test solution cannot be filtered according to point 5.1.7, appropriate methods should be taken in order to obtain a clear solution. (2) Where an inhibition of enzymes occurs, it is advisable to apply a method which involves the addition of known amounts of pure starch. ( 1 ) U is the international unit of enzyme activity.