UGANDA STANDARD  
    US ISO 14902
First Edition
   2009 -09-04
 
Reference numbe r 
   US ISO 14902:2001
 © UNBS 200 9 
  
Animal feeding stuffs — Determination of trypsin inhibitor 
activity of soya products 
 US ISO 14902:2001  
 
© UNBS  2009 – All rights reserved Compliance with this standard does not, of itself confer immunity from legal obligations 
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct application  
© UNBS 2009  
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E-mail: unbs@infocom.co.ug
     US ISO 14902: 2001  
© UNBS  2009 - All rights reserved National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry  of Tourism, Trade and 
Industry established by the Act of Parliament of 198 3, of the Laws of Uganda.  UNBS is mandated to co-
ordinate the elaboration of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex A limentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT/SPS Agr eements of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a gi ven field or area and consists of representatives of 
consumers, traders, academician s, manufacturers, governme nt and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committ ee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council. 
This Uganda Standard, US ISO 14902:2001,  Animal feeding stuffs — Determination of trypsin inhibitor activity
of soya products, is identical with and  has been reproduced from an International Standard ISO 14902:
2001, Animal feeding stuffs — Determination of tr ypsin inhibitor activity of soya products, and has been
adopted as a Uganda Standard . 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." Reference number
ISO14902:2001(E)
©ISO2001INTERNATIONAL
STANDARDISO
14902
Firstedition
2001-10-15
Animal feedingstuffs— Determinationof
trypsininhibitor activityof soya products
Aliments des animaux — Dosage de l'activitédes inhibiteurs trypsiques
des produits desojaISO 14902:2001(E)
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ii © ISO2001 –All rights reservedISO 14902:2001(E)
© ISO2001 –All rights reserved iiiContents Page
Foreword....................................................................................................................... ..............................................iv
1 Scope......................................................................................................................... .....................................2
2 Normativereference............................................................................................................ ..........................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 Numberof determinations...................................................................................................... ......................4
9.2 Sample extraction............................................................................................................ ..............................4
9.3 Dilution of sample extract..................................................................................................... ........................5
9.4 Measurement of trypsin activityof working solution............................................................................... .5
9.5 Measurement of trypsin inhibitoractivity....................................................................................... ............5
10 Calculation.................................................................................................................. ....................................6
10.1 Inhibition percentageof sampleextract solutions............................................................................... ......6
10.2 Trypsin inhibitor activity................................................................................................... ............................7
11 Precision.................................................................................................................... .....................................7
11.1 Interlaboratorytests....................................................................................................... ...............................7
11.2 Repeatability.............................................................................................................. .....................................7
11.3 Reproducibility............................................................................................................ ...................................8
12 Test report................................................................................................................... ...................................8
Annex A (normative) Dilution schemeforsample extract......................................................................................9
Annex B (informative) Resultsof interlaboratorytest...........................................................................................11
Bibliography................................................................................................................... ...........................................12ISO 14902:2001(E)
iv © ISO2001 –All rights reservedForeword
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 hasthe right to be represented on that committee. International organizations, governmental and non-governmental, inliaison with ISO, also take part in the work. ISO collaborates closely with the International ElectrotechnicalCommission(IEC) onall matters of electrotechnicalstandardization.
InternationalStandards are drafted in accordance with the rules givenintheISO/IEC Directives, Part3.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publicationas anInternationalStandardrequires approval byat least 75% of themember 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 beheldresponsiblefor identifyinganyor allsuch patent rights.
International Standard ISO14902 was prepared by Technical Committee ISO/TC 34, Food products ,
SubcommitteeSC 10, Animalfeedingstuffs .
Annex Aforms anormativepart of this International Standard. AnnexB is for informationonly.INTERNATIONAL STANDARD ISO 14902:2001(E)
© ISO2001 –All rights reserved 1Animalfeedingstuffs —Determinationoftrypsininhibitor activity
ofsoya products
1 Scope
This International Standard specifies a method for the determination of the trypsin inhibitor activity (TIA) of soya
products.
This trypsin inhibitor activityis indicative of the degree of toasting of these products.Thedetection limit of themethod is 0,5mg/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 thesepublications 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 IECmaintain registers of currentlyvalid InternationalStandards.
ISO3696:1987, Water for analytical laboratory use —Specificationandtest methods
3 Term and definition
For the purposes of this International Standard, thefollowing term anddefinition 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 inhibitoractivityisexpressed inmilligrams pergram.
4P r i n c i p l e
Trypsin inhibitors are extracted from thesampleat pH 9,5.
The remaining trypsin activity is measured by adding benzoyl- L-arginine- p-nitroanilide (L-BAPA) as substrate. The
quantityof released p-nitroaniline is measuredspectrometrically.ISO 14902:2001(E)
2 © ISO2001 –All rights reserved5 Reagents andmaterials
Useonlyreagents of recognized analyticalgrade.
5.1 Water ,complying with at least grade3 inaccordance with ISO3696.
5.2 Sodiumhydroxidesolution ,c(NaOH)=0,01mol/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)=0,1mol/l.
5.6 Hydrochloric acid ,c(HCl)=0,001mol/l.
5.7 Acetic acid ,c(CH3COOH) =5,3mol/l.
5.8 Calcium chloridedihydrate ,Ca Cl2/Gd72H2O.
5.9 Calcium chloridesolution inhydrochloric acid.
Dissolve 735mg of calcium chloride dihydrate (5.8) in 1l of hydrochloric acid (5.6) and check the pH. The pH shall
be3,0 /Gb10,1.
5.10 Bovinetrypsin (Merck No. 24579or equivalent).1)
See 9.4for measurement of theactivity. Store in the refrigerator (6.3).
5.11 Trypsin stocksolution
Allow the trypsin (5.10) to reach room temperature. Dissolve 27,0mg of trypsin in the calcium chloride solution
(5.9) in a100ml volumetric flask (6.1) and diluteto themark withthecalcium chloride solution. This solution can beusedfor 5days at most whenstored in the refrigerator (6.3).
5.12 Trypsin working solution .
Pipette 5ml of the trypsin stock solution (5.11) into a 100ml 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 Dimethylsulfoxide(DMSO) .
5.16 Trisbuffer/calcium chloridesolution .
Dissolve 6,05g of Tris (5.14) and 735mg of calcium chloride (5.8) in 900ml of water in a 1l graduated measuring
cylinder. Adjust the pH to 8,2 /Gb10,1 with hydrochloric acid(5.3) anddiluteto1l with water.
5.17 L-BAPAreagent .
Prepare this reagent on the day of use. Dissolve 60mg of L-BAPA (5.13) in 1ml of DMSO (5.15) in a 100ml
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 usersof thisInternational Standardanddoes not constitute an endorsement byISOofthisproduct.ISO 14902:2001(E)
© ISO2001 –All rights reserved 36 Apparatus
Usual laboratoryapparatus and, in particular, thefollowing.
6.1 Volumetricflasks , of capacity100ml.
6.2 Cuvettes ,withopticalpathlength10mm.
6.3 Refrigerator , controlledat a temperature of (4 /Gb13)°C.
6.4 pH-meter , with an inaccuracyof 0,05units.
6.5 Test tubemixer .
6.6 Spectrometer , suitablefor measurements at a wavelengthof 410nm.
6.7 Stopwatch .
6.8 Waterbath , withcirculation pump, capable of beingmaintainedat (37 /Gb10,25)°C.
6.9 Grindingapparatus ,p r ov i d edw i t ha0 ,5mms i e v e .
6.10 Centrifuge , operatingat aradialacceleration of approximately1500 gn.
6.11 Centrifugetubes .
7 Sampling
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changedduringtransport 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 Preparationoftestsample
Usingthe grindingapparatus (6.9), grindarepresentative part of sample so that heat production is minimal. Mix thegroundsamplethoroughly.
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 repeatabilityconditions.
9.2 Sample extraction
Weigh 1g /Gb10,001 g of the prepared test sample (clause8) in a 100ml conical flask and add 50ml of sodium
hydroxide solution (5.2). Completelysuspend the sample. Adjust the pH to 9,5 /Gb10,1 with hydrochloric acid (5.4 and
5.5). Rinsetheelectrode with as little water as possible. Close theconicalflask and store overnight (15h to 24h) intherefrigerator (6.3). Placeintherefrigerator thequantityof water neededfor makingupthesampleextracts.ISO 14902:2001(E)
4 © ISO2001 –All rights reservedTransfer thesampleextract to a100mlvolumetric flask (6.1), dilutetothe mark with water from the refrigerator and
mix. Store the volumetric flask in the refrigerator. The sample extract remains stable for one day. Aftersedimentation for 15min, the sample extract may be worked up further and diluted as required. Dilutions dependontheexpectedTIA valueof thesampleandarecarried out with water at room temperature.
9.3 Dilution of sampleextract
Estimate the TIA value of the sample and prepare three different dilutions of the sample extract on the basis of thedilution scheme in TableA.1, so that it may be expected that as a result of the TIA measurement (9.5) at least one
of thethreeinhibitionpercentages obtained will be within the rangeof 40% to 60%.
If noneof thethreeresults is withinthis range, the estimationshouldbeadapted and the procedurerepeated.
9.4 Measurementoftrypsin activityofworking 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/G2dAbr) should be 0,380 /Gb10,050. In this is not the case, check the qualiity
of thetrypsin(5.10). If necessary, takeafreshjar of trypsin.
Pipette intocentrifugetubes accordingto thefollowingscheme:
Blank standard Standard
ml ml
L-BAPAreagent (5.17) 5 5
Water(5.1) 3 3
Acetic acid (5.7) 1 0
Mix the contents of the tubes with the test tube mixer (6.5) and place the tubes in the water bath (6.8) for 10min.
Add:
Blank standard Standard
ml ml
Trypsin working solution(5.12) 1 1
Mix the contents of the tubes with the test tube mixer and place the centrifuge tubes back in the water bath. After
10min /Gb15s of incubation, add thefollowing:
Blank standard Standard
ml ml
Acetic acid (5.7) 0 1
Mix thecontents of the tubes withthetest tubemixer.
Centrifugethetubes for 10min inthecentrifuge(6.10) at aradial accelerationof approximately1500 gn.
Measure the absorbance of the clear solutions relative to water in the spectrometer (6.6) at 410nm in a 10mm
cuvette(6.2).
These solutions remain stable for at least 2h.ISO 14902:2001(E)
© ISO2001 –All rights reserved 59.5 Measurementoftrypsin inhibitor activity
Pipette intocentrifugetubes accordingto thefollowingscheme.
Prepare for each dilution of sample extract (9.3) a corresponding blank solution. Sample extract solutions and
corresponding blank solutions shall be dealt withsimultaneouslyintheprocedure, including centrifuging.
Blank standard Standard Blank sample Sample
ml ml ml ml
L-BAPAreagent (5.17) 5 5 5 5
Diluted sample extract (9.3) 0 0 1 1
Water(5.1) 3 3 2 2
Acetic acid (5.7) 1 0 1 0
Mix the contents of the tubes with the test tube mixer (6.5) and place the tubes in the water bath (6.8) for 10min.Add the following:
Blank standard Standard Blank sample Sample
ml ml ml ml
Trypsin working solution(5.12) 1 1 1 1
Mix the contents of the tubes with the test tube mixer and place the centrifuge tubes back in the water bath (6.8).
After 10min /Gb15s incubationadd thefollowing:
Blank standard Standard Blank sample Sample
ml ml ml ml
Acetic acid (5.7) 0 1 0 1
Mix thecontents of the tubes withthetest tubemixer.
Centrifugethetubes for 10min inthecentrifuge(6.10) at aradial accelerationof approximately1500 gn.
Measure the absorbance of the clear solutions relative to water in the spectrometer (6.6) at 410nm in a 10mm
cuvette(6.2).
These solutions remain stable for at least 2h.
10 Calculation
10.1 Inhibition percentageofsample extractsolutions
Calculate the inhibitionpercentageof the sampleextract solutions bytheequation:
/G28/G29 /G28 /G29
/G28/G29rb r sb s
rb r100%AA AAi
AA/G2d/G2d /G2d/G3d/Gb4
/G2dISO 14902:2001(E)
6 © ISO2001 –All rights reservedwhere
iis the inhibition percentage, in percent;
Aris the absorbanceof thesolution withstandard;
Abris the absorbanceof theblank withstandard;
Asis the absorbanceof thesolution withsample;
Absis the absorbanceof theblank withsample.
10.2 Trypsininhibitor activity
Calculate the trypsin inhibitor activity, expressed in milligrams of inhibited trypsin per gram of sample, by the
equation:
112
0TIA100%mffi
m/G3d/Gb4
where
TIA is the trypsininhibitor activity, inmilligrams per gram;
iis the inhibition percentage, inpercent;
m0is themass of thetest sample, ingrams;
m1is themass of trypsin, inmilligrams;
f1is the dilution of the sample extract [(100ml /Gb4100ml)/V,w h e r eVis the volume derived from TableA.1,
inmillilitres];
f2is a conversion factor (2,8 /Gb410/G2d4) based on the purity of trypsin (56%, see refs. [1] and [2]) and on the
dilution of trypsin accordingto5.11and 5.12.
Roundtheresultto the nearest0,1mg/g.
11 Precision
11.1 Interlaboratorytests
Details of interlaboratory tests on the precision of the method are given in annexB. The values derived from these
tests maynotbe applicabletoconcentrationranges andmatrices other thanthosegiven.
11.2 Repeatability
The absolute difference between two independent single test results, obtained using the same method on identical
test material in the same laboratory by the same operator using the same equipment within a short interval of time,
willinnot morethan 5% of cases exceedtherepeatabilitylimit ( r) mentioned in or derivedfrom Table1.ISO 14902:2001(E)
© ISO2001 –All rights reserved 7Table1—Repeatabilitylimit (r)and reproducibilitylimit (R)
Sample Trypsin inhibitoractivity rR
mg/g mg/g mg/g
Soya 1 1,53 0,18 1,11
Soya 2 1,30 0,09 2,11
Toastedsoya beans 2,08 1,03 1,88
11.3 Reproducibility
The absolute difference between two single test results, obtained using the same method on identical test material
in different laboratories by different operators using different equipment, will in not more than 5% of cases exceed
the reproducibilitylimit ( R) mentioned in or derivedfrom Table1.
12 Testreport
Thetest report shallspecify:
/Gbeall information necessaryfor the complete identification of the sample;
/Gbethesamplingmethodused, if known;
/Gbethetest methodused, withreferencetothis International Standard;
/Gbeall operating details not specified in this International Standard, or regarded as optional, together with details of
anyincidents whichmayhave influenced the test results;
/Gbethetest result obtained or, if the repeatabilityhas beenchecked, thetwotest results obtained.ISO 14902:2001(E)
8 © ISO2001 –All rights reservedAnnex A
(normative)
Dilutionschemefor sampleextract
TableA.1 gives the dilution scheme to be followed. FiguresA.1 and A.2 show an example of a graphical
representationof the dilution scheme.
TableA.1 —Dilution scheme
Theoretical dilutionatdifferentinhibition percentages
ml/100 gExpected TIA
mg/g
40 % 50 % 60 %
0,5 61 76 91
1,0 30 38 45
1,5 20 25 30
2,0 15 19 23
2,5 12 15 18
3,0 10 13 15
3,5 8,6 11 13
4,0 7,6 9,5 11
4,5 6,7 8,4 10
5,0 6,0 7,6 9,1
6 5,0 6,3 7,6
7 4,3 5,4 6,5
8 3,8 4,7 5,7
9 3,4 4,2 5,0
10 3,0 3,8 4,5
11 2,7 3,4 4,1
12 2,5 3,2 3,8
13 2,3 2,9 3,5
14 2,2 2,7 3,2
15 2,0 2,5 3,0
16 1,9 2,4 2,8
17 1,8 2,2 2,7
18 1,7 2,1 2,5
19 1,6 2,0 2,4
20 1,5 1,9 2,3
25 1,2 1,5 1,8ISO 14902:2001(E)
© ISO2001 –All rights reserved 9Figure A.1 —Relationship between trypsin inhibitoractivityand theoreticaldilution of thesample
Figure A.2 —Detailof Figure A.1ISO 14902:2001(E)
10 © ISO2001 –All rights reservedAnnex B
(informative)
Results ofinterlaboratorytest
The precision of the method was established in 1998 by interlaboratory tests carried out in accordance with
ISO 5725-1 [3] and ISO 5725-2 [4] for the determination of the reproducibility. For the determination of the
repeatability limit, seven laboratories carried out duplicate analyses of samples of soya. For the determination ofthe reproducibility limit, single analyses of two samples soya and one sample of toasted soya beans were carriedout bysevenlaboratories.
TableB.1 —Statisticalresultsof interlaboratorytests
Samplea
Parameter
123
Numberof laboratories 7 7 7
Numberof laboratories retainedaftereliminating
outliers777
Mean trypsin inhibitoractivity, mg/g 1,30 1,53 1,88
Repeatabilitystandard deviation ( sr),mg/g 0,03 0,06 0,37
Repeatabilitycoefficient ofvariation,% 2,31 4,18 1,03
Repeatabilitylimit ( r)[r=2 , 8 /Gb4sr],mg/g 0,09 0,18 1,03
Reproducibilitystandarddeviation ( sR), mg/g 0,75 0,40 0,67
Reproducibilitycoefficient of variation, % 57,7 25,9 35,7
Reproducibilitylimit( R)[R=2 , 8 /Gb4sR],mg/g 2,11 1,11 1,88
aSamples 1 and2: soya.
Sample 3: toastedsoya beans.ISO 14902:2001(E)
© ISO2001 –All rights reserved 11Bibliography
[1] K AKADEM. L., S IMONSN., LIENERI. E. An Evaluation of Natural vs. synthetic Substrates for Measuring the
Antitryptic Activityof Soybean Samples. CerealChem. ,46, 1969, pp. 518-526
[2] S MITHC., VANMEGENW.,TWAALFHOVEN L., HITCHCOCK C. The Determination of Trypsin Inhibitor Levels in
Foodstuffs. J.Sci.Food Agric. ,31, 1980, pp. 341-350
[3] ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results —Part1:
General principles and definitions
[4] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results —Part2: Basic
methodfor the determination of repeatability andreproducibility of a standardmeasurement method
[5] ISO 6497, Animalfeedingstuffs —Sampling
[6] ISO 6498:1983, Animalfeedingstuffs —Preparationof test sampleISO 14902:2001(E)
ICS 65.120
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