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1、Reference number ISO 14902:2001(E) ISO 2001 INTERNATIONAL STANDARD ISO 14902 First edition 2001-10-15 Animal feeding stuffs Determination of trypsin inhibitor activity of soya products Aliments des animaux Dosage de lactivit des inhibiteurs trypsiques des produits de soja Copyright International Org
2、anization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:46:44 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14902:2001(E) PDF disclaimer This PDF file may contain embedded typefa
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5、zed for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below. ISO 2001 All rights reserved. Unless otherwise specified, no p
6、art of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale
7、 56 ? CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.ch Web www.iso.ch Printed in Switzerland ii ISO 2001 All rights reserved Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/997254
8、5001 Not for Resale, 04/21/2007 01:46:44 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14902:2001(E) ISO 2001 All rights reservediii ContentsPage Foreword.iv 1Scope 2 2Normative reference2 3Term and definition .2 4Principle2 5Reagents and materials 2 6Apparatus.3 7Sam
9、pling.4 8Preparation of test sample4 9Procedure.4 9.1Number of determinations4 9.2Sample extraction4 9.3Dilution of sample extract.5 9.4Measurement of trypsin activity of working solution 5 9.5Measurement of trypsin inhibitor activity.5 10Calculation6 10.1Inhibition percentage of sample extract solu
10、tions.6 10.2Trypsin inhibitor activity.7 11Precision.7 11.1Interlaboratory tests7 11.2Repeatability.7 11.3Reproducibility.8 12Test report8 Annex A (normative) Dilution scheme for sample extract9 Annex B (informative) Results of interlaboratory test.11 Bibliography12 Copyright International Organizat
11、ion for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:46:44 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14902:2001(E) iv ISO 2001 All rights reserved Foreword ISO (the Internationa
12、l Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been es
13、tablished has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardi
14、zation. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 7
15、5 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. InternationalStandardISO 14902waspreparedbyTec
16、hnicalCommitteeISO/TC 34,Foodproducts, Subcommittee SC 10, Animal feeding stuffs. Annex A forms a normative part of this International Standard. Annex B is for information only. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical St
17、andards 1/9972545001 Not for Resale, 04/21/2007 01:46:44 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARDISO 14902:2001(E) ISO 2001 All rights reserved1 Animal feeding stuffs Determination of trypsin inhibitor activity of soya products 1Scope This Inte
18、rnational Standard specifies a method for the determination of the trypsin inhibitor activity (TIA) of soya products. This trypsin inhibitor activity is indicative of the degree of toasting of these products. The detection limit of the method is 0,5 mg/g. 2Normative reference The following normative
19、 document contains provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standard are
20、encouraged to investigate the possibility of applying the most recent edition of the normative document indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO
21、 3696:1987, Water for analytical laboratory use Specification and test methods 3Term and definition For the purposes of this International Standard, the following term and definition applies. 3.1 trypsin inhibitor activity TIA mass of trypsin inhibited by the procedure described in this Internationa
22、l Standard, divided by the mass of the test sample NOTEThe trypsin inhibitor activity is expressed in milligrams per gram. 4Principle Trypsin inhibitors are extracted from the sample at pH 9,5. The remaining trypsin activity is measured by adding benzoyl-L-arginine-p-nitroanilide (L-BAPA) as substra
23、te. The quantity of released p-nitroaniline is measured spectrometrically. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:46:44 MDTNo reproduction or networking permitted wit
24、hout license from IHS -,-,- ISO 14902:2001(E) 2 ISO 2001 All rights reserved 5Reagents and materials Use only reagents of recognized analytical grade. 5.1Water, complying with at least grade 3 in accordance with ISO 3696. 5.2Sodium hydroxide solution, c(NaOH) = 0,01 mol/l. 5.3Hydrochloric acid, c(HC
25、l) = 6 mol/l. 5.4Hydrochloric acid, c(HCl) = 1 mol/l. 5.5Hydrochloric acid, c(HCl) = 0,1 mol/l. 5.6Hydrochloric acid, c(HCl) = 0,001 mol/l. 5.7Acetic acid, c(CH3COOH) = 5,3 mol/l. 5.8Calcium chloride dihydrate, CaCl2?2H2O. 5.9Calcium chloride solution in hydrochloric acid. Dissolve 735 mg of calcium
26、 chloride dihydrate (5.8) in 1 l of hydrochloric acid (5.6) and check the pH. The pH shall be 3,0?0,1. 5.10Bovine trypsin (Merck No. 24579 or equivalent).1) See 9.4 for measurement of the activity. Store in the refrigerator (6.3). 5.11Trypsin stock solution Allow the trypsin (5.10) to reach room tem
27、perature. Dissolve 27,0 mg of trypsin in the calcium chloride solution (5.9) in a 100 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). 5.12Trypsin working solution. Pipette 5 ml of
28、 the trypsin stock solution (5.11) into a 100 ml volumetric flask (6.1) and dilute to the mark with calcium chloride solution (5.9). 5.13Benzoyl-L-arginine-p-nitroanilide (L-BAPA). 5.14Tris-(hydroxymethyl)aminomethane (Tris). 5.15Dimethyl sulfoxide (DMSO). 5.16Tris buffer/calcium chloride solution.
29、Dissolve 6,05 g of Tris (5.14) 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. 5.17L-BAPA reagent. Prepare this reagent on the day of use. Dissolve 60 mg of L-BAPA (5.13
30、) in 1 ml of DMSO (5.15) in a 100 ml volumetric flask (6.1) and dilute to the mark with Tris buffer/calcium chloride solution (5.16). 1)Merck No. 24579 is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard an
31、d does not constitute an endorsement by ISO of this product. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:46:44 MDTNo reproduction or networking permitted without license f
32、rom IHS -,-,- ISO 14902:2001(E) ISO 2001 All rights reserved3 6Apparatus Usual laboratory apparatus and, in particular, the following. 6.1Volumetric flasks, of capacity 100 ml. 6.2Cuvettes, with optical path length 10 mm. 6.3Refrigerator, controlled at a temperature of (4?3) C. 6.4pH-meter, with an
33、inaccuracy of 0,05 units. 6.5Test tube mixer. 6.6Spectrometer, suitable for measurements at a wavelength of 410 nm. 6.7Stopwatch. 6.8Water bath, with circulation pump, capable of being maintained at (37?0,25) C. 6.9Grinding apparatus, provided with a 0,5 mm sieve. 6.10Centrifuge, operating at a radi
34、al acceleration of approximately 1 500 gn. 6.11Centrifuge tubes. 7Sampling 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.
35、A recommended sampling method is given in ISO 6497 5. 8Preparation 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. 9Procedure 9.1Number of determinations If it is required to check whether
36、the repeatability limit (11.2) is met, carry out two single determinations in accordance with 9.2 and 9.5 under repeatability conditions. 9.2Sample extraction Weigh 1 g?0,001 g of the prepared test sample (clause 8) in a 100 ml conical flask and add 50 ml of sodium hydroxide solution (5.2). Complete
37、ly 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 (15 h to 24 h) in the refrigerator (6.3). Place in the refrigerator the quantity of water needed for making up the s
38、ample extracts. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:46:44 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14902:2001(E) 4 ISO 2001 Al
39、l rights reserved Transfer the sample extract to a 100 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 15 min, the sample extract may be work
40、ed 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. 9.3Dilution of sample extract Estimate the TIA value of the sample and prepare three different dilutions of the sample extract on the basis of the dilut
41、ion 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
42、repeated. 9.4Measurement of trypsin activity of working solution Check the activity of each batch of trypsin (5.10). The difference between the absorbance of the working solution (5.12) and the absorbance of the blank (Ar?Abr) should be 0,380?0,050. In this is not the case, check the qualiity of the
43、 trypsin (5.10). If necessary, take a fresh jar of trypsin. Pipette into centrifuge tubes according to the following scheme: Blank standardStandard mlml L-BAPA reagent (5.17)55 Water (5.1)33 Acetic acid (5.7)10 Mix the contents of the tubes with the test tube mixer (6.5) and place the tubes in the w
44、ater bath (6.8) for 10 min. Add: Blank standardStandard mlml Trypsin working solution (5.12)11 Mix the contents of the tubes with the test tube mixer and place the centrifuge tubes back in the water bath. After 10 min?5 s of incubation, add the following: Blank standardStandard mlml Acetic acid (5.7
45、)01 Mix the contents of the tubes with the test tube mixer. Centrifuge the tubes for 10 min in the centrifuge (6.10) at a radial acceleration of approximately 1 500 gn. Measure the absorbance of the clear solutions relative to water in the spectrometer (6.6) at 410 nm in a 10 mm cuvette (6.2). These
46、 solutions remain stable for at least 2 h. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:46:44 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO
47、14902:2001(E) ISO 2001 All rights reserved5 9.5Measurement of trypsin inhibitor activity Pipette into centrifuge tubes according to the following scheme. Prepare for each dilution of sample extract (9.3) a corresponding blank solution. Sample extract solutions and corresponding blank solutions shall
48、 be dealt with simultaneously in the procedure, including centrifuging. Blank standardStandardBlank sampleSample mlmlmlml L-BAPA reagent (5.17)5555 Diluted sample extract (9.3)0011 Water (5.1)3322 Acetic acid (5.7)1010 Mix the contents of the tubes with the test tube mixer (6.5) and place the tubes in the water bath (6.8) for 10 min. Add the following: Blank standardStandardBlank sampleSample mlmlmlml Trypsin working solution (5.12)1111 Mix the contents of the tubes with the test tube mixer and