BS EN ISO 14902:2001 pdf – Animal feeding stuffs — Determination of trypsin inhibitor activity of soya products.
5 Reagents and materials Use only reagents of recognized analytical grade. 5.1 Water, complying with at least grade 3 in accordance with ISO 3696. 5.2 Sodium hydroxide solution, c(NaOH) = 0,01 mol/l. 5.3 Hydrochloric acid, c(HCl) = 6 mol/l. 5.4 Hydrochloric acid, c(HCl) = 1 mol/l. 5.5 Hydrochloric acid, c(HCl) = 0,1 mol/l. 5.6 Hydrochloric acid, c(HCl) = 0,001 mol/l. 5.7 Acetic acid, c(CH 3 COOH) = 5,3 mol/l. 5.8 Calcium chloride dihydrate, CaCl 2 ? 2H 2 O. 5.9 Calcium chloride solution in hydrochloric acid. Dissolve 735 mg of calcium chloride dihydrate (5.8) in 1 l of hydrochloric acid (5.6) and check the pH. The pH shall be 3,0 ? 0,1 . 5.10 Bovine trypsin (Merck No. 24579 or equivalent). 1 ) See 9.4 for measurement of the activity. Store in the refrigerator (6.3). 5.11 Trypsin stock solution Allow the trypsin (5.1 0) to reach room temperature. Dissolve 27,0 mg of trypsin in the calcium chloride solution (5.9) in a 1 00 ml volumetric flask (6.1 ) and dilute to the mark with the calcium chloride solution. This solution can be used for 5 days at most when stored in the refrigerator (6.3). 5.12 Trypsin working solution. Pipette 5 ml of the trypsin stock solution (5.1 1 ) into a 1 00 ml volumetric flask (6.1 ) and dilute to the mark with calcium chloride solution (5.9). 5.13 Benzoyl- L -arginine-p-nitroanilide (L-BAPA). 5.14 Tris-(hydroxymethyl)aminomethane (Tris). 5.15 Dimethyl sulfoxide (DMSO). 5.16 Tris buffer/calcium chloride solution. Dissolve 6,05 g of Tris (5.1 4) and 735 mg of calcium chloride (5.8) in 900 ml of water in a 1 l graduated measuring cylinder. Adjust the pH to 8,2 ? 0,1 with hydrochloric acid (5.3) and dilute to 1 l with water. 5.17 L-BAPA reagent. Prepare this reagent on the day of use. Dissolve 60 mg of L-BAPA (5.1 3) in 1 ml of DMSO (5.1 5) in a 1 00 ml volumetric flask (6.1 ) and dilute to the mark with Tris buffer/calcium chloride solution (5.1 6).
6 Apparatus Usual laboratory apparatus and, in particular, the following. 6.1 Volumetric flasks, of capacity 1 00 ml. 6.2 Cuvettes, with optical path length 1 0 mm. 6.3 Refrigerator, controlled at a temperature of (4 ? 3) °C. 6.4 pH-meter, with an inaccuracy of 0,05 units. 6.5 Test tube mixer. 6.6 Spectrometer, suitable for measurements at a wavelength of 41 0 nm. 6.7 Stopwatch. 6.8 Water bath, with circulation pump, capable of being maintained at (37 ? 0,25) °C. 6.9 Grinding apparatus, provided with a 0,5 mm sieve. 6.10 Centrifuge, operating at a radial acceleration of approximately 1 500 g n . 6.11 Centrifuge tubes. 7 Sampling It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 6497 . 8 Preparation of test sample Using the grinding apparatus (6.9), grind a representative part of sample so that heat production is minimal. Mix the ground sample thoroughly. 9 Procedure 9.1 Number of determinations If it is required to check whether the repeatability limit (1 1 .2) is met, carry out two single determinations in accordance with 9.2 and 9.5 under repeatability conditions. 9.2 Sample extraction Weigh 1 g ? 0,001 g of the prepared test sample (clause 8) in a 1 00 ml conical flask and add 50 ml of sodium hydroxide solution (5.2). Completely suspend the sample. Adjust the pH to 9,5 ? 0,1 with hydrochloric acid (5.4 and 5.5). Rinse the electrode with as little water as possible. Close the conical flask and store overnight (1 5 h to 24 h) in the refrigerator (6.3). Place in the refrigerator the quantity of water needed for making up the sample extracts.
Transfer the sample extract to a 1 00 ml volumetric flask (6.1 ), dilute to the mark with water from the refrigerator and mix. Store the volumetric flask in the refrigerator. The sample extract remains stable for one day. After sedimentation for 1 5 min, the sample extract may be worked up further and diluted as required. Dilutions depend on the expected TIA value of the sample and are carried out with water at room temperature. 9.3 Dilution of sample extract Estimate the TIA value of the sample and prepare three different dilutions of the sample extract on the basis of the dilution scheme in Table A.1 , so that it may be expected that as a result of the TIA measurement (9.5) at least one of the three inhibition percentages obtained will be within the range of 40 % to 60 %. If none of the three results is within this range, the estimation should be adapted and the procedure repeated. 9.4 Measurement of trypsin activity of working solution Check the activity of each batch of trypsin (5.1 0). The difference between the absorbance of the working solution (5.1 2) and the absorbance of the blank (A r ? A br ) should be 0,380 ? 0,050. In this is not the case, check the qualiity of the trypsin (5.1 0). If necessary, take a fresh jar of trypsin.