ISO 14902:2001

INTERNATIONAL ISO
STANDARD 14902
First edition
2001-10-15
Animal feeding stuffs — Determination of
trypsin inhibitor activity of soya products
Aliments des animaux — Dosage de l'activité des inhibiteurs trypsiques
des produits de soja
Reference number
©
ISO 2001
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ii © ISO 2001 – All rights reserved

Contents Page
Foreword.iv
1 Scope .2
2 Normative reference .2
3 Term and definition .2
4 Principle.2
5 Reagents and materials .2
6 Apparatus .3
7 Sampling.4
8 Preparation of test sample.4
9 Procedure .4
9.1 Number of determinations .4
9.2 Sample extraction.4
9.3 Dilution of sample extract.5
9.4 Measurement of trypsin activity of working solution .5
9.5 Measurement of trypsin inhibitor activity .5
10 Calculation.6
10.1 Inhibition percentage of sample extract solutions.6
10.2 Trypsin inhibitor activity.7
11 Precision.7
11.1 Interlaboratory tests .7
11.2 Repeatability.7
11.3 Reproducibility.8
12 Test report .8
Annex A (normative) Dilution scheme for sample extract.9
Annex B (informative) Results of interlaboratory test.11
Bibliography.12
Foreword
ISO (the International 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 established 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 standardization.
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 75 % 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.
International Standard ISO 14902 was prepared by Technical Committee ISO/TC 34, Food products,
Subcommittee SC 10, Animal feeding stuffs.
Annex A forms a normative part of this International Standard. Annex B is for information only.
iv © ISO 2001 – All rights reserved

INTERNATIONAL STANDARD ISO 14902:2001(E)
Animal feeding stuffs — Determination of trypsin inhibitor activity
of soya products
1 Scope
This International 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.
2 Normative reference
The following normative 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 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 3696:1987, Water for analytical laboratory use — Specification and test methods
3 Term 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 International Standard, divided by the mass of the test
sample
NOTE The 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 substrate. The
quantity of released p-nitroaniline is measured spectrometrically.
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 COOH) = 5,3 mol/l.
5.8 Calcium chloride dihydrate,CaCl �2H O.
2 2
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.
1)
5.10 Bovine trypsin (Merck No. 24579 or equivalent).
See 9.4 for measurement of the activity. Store in the refrigerator (6.3).
5.11 Trypsin stock solution
Allow the trypsin (5.10) to reach room temperature. 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).
5.12 Trypsin working solution.
Pipette 5 ml of 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).
5.13 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 (5.14) 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.
5.17 L-BAPA reagent.
Prepare this reagent on the day of use. Dissolve 60 mg of L-BAPA (5.13) 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 and does not constitute an endorsement by ISO of this product.
2 © ISO 2001 – All rights reserved

6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 Volumetric flasks, of capacity 100 ml.
6.2 Cuvettes, with optical path length 10 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 410 nm.
6.7 Stopwatch.
6.8 Water bath, with circulation pump, capable of being maintained at (37 � 0,25) °C.
6.9 Grinding apparatus,providedwitha0,5mm 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 [5].
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 (11.2) 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 100 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 (15 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 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 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 (5.10). The difference between the absorbance of the working solution
(5.12) and the absorbance of the blank (A � A ) should be 0,380 � 0,050. In this is not the case, check the qualiity
r br
of the trypsin (5.10). If necessary, take a fresh jar of trypsin.
Pipette into centrifuge tubes according to the following scheme:
Blank standard Standard
ml ml
L-BAPA reagent (5.17) 5 5
Water (5.1) 3 3
Acetic acid (5
...