ISMSO 14902: 2001 indian Standard ANIMAL FEEDING STUFFS — DETERMINATION OF TRYPSIN INHIBITOR ACTIVITY OF SOYA PRODUCTS 1 Scope ‘This Intemational Standard specifies a msthod for the determination of the Urypsin inhibitor acivily (TIA) of soya products. ‘This ’ypsin inhibitor activity ie indicative of the dagroa of taas:ing of these products. ‘The detection iimil of he method is 0,5 ma. 2 Normative reference The following hormotive document coniains provisions which. through reference in this text, conaltute provisions of thas International Standard, Por dated referaneas, subsequers amendments to, of revisions of, any of these publications do not apply. However, parfes Ia egraementa based on this International Standard are encouraged to Investigate the porsleilly oF sppiying the most recent edition of Ihe normalive documant indicated below. For vedated references, the latest ecition of the normativa document relsrrad to applies. Members of ISO and IEG mointain registers of curently vale Inlernationel Stondards, 1S0 3696-1967, Watar for analytical laboretiry vse — Specification end fest methods 3° Term and definition For the purposes of this Intarnational Standard, the following ferm end definition applcs. a4 {rypain inhibitor activity TA ‘mass of trypsin inhibited by the procedure sample cribed in this Entematlonal Slandard, divised by the mass of the test NOTE The trypsin Inniblor activity it exprosted In miligrame por gram. 4 Principle Trypsin inhibitors are extracted from the samplo at pH 9.5. The remaining trypsin activity is measured by adding benzoyt-arginine-p-nitroaniiide (L-BAPA) os substrate. The ‘quantty of released p-nitoaniline is measured spactrometically,ISSO 14902: 2001 5 Reagents and materials Use only reagents of recognized analyécal grade, $1. Water, complying with al least grade 3 in accordance with ISO 3696. 52. Sodium hydroxide solution, c(MaOH) = 0.01 mot 5.3 Hydrochloric acid, (HC!) = 6 mou. 54. Hydrochloric acid, HCl) = ¥ mout 5.8 Hydrochlorle acld, (HC!) = 0,1 molt, 5.4 Hydrochiorie ack, HCl) = 0,004 moi 5.7 Acetic ackd, e{CHsCOOH) = 5.3 mot). 5.4 Calcium chloride dihydrate, CaCiy 2120. 5.9 Calclum chloride solution in hysrochione seis Dissolve 738 mg of caichum chloride dimydraia (8.8) kn 1 of hydrechloric acid (8.6) and chock the pH. The pH shall be 3020, 5.10 Bovine trypaln (Merck No, 24579 or equivalent”) See 9.4 for measurement of the activty. Store in the refrigerator (6.3). 5.11 Trypsin stock sotution ‘Allow tho tryptin (5.10) 4o foach room temperature. Dissolve 27,0 mg of trypsin In the cakivm chloride roliion (5.8) ina 100 mi volumetric flask (6.1) and diute to the mark wath the calcium chiorde sotution. This solution can be Used for 5 days at most when stored in the retnigorator (6.3). 5.12. Trypain working solution, Prpate § mi of the typain a sacha chloride sohaton(! 3.43. Benzoyl-arginine-p-nitroanilide (L-BAPAY. 514. Trie-{hydroxymethyf}aminomethane (Tris). 5.15 Dimathy! sulfoxide (0M SO} olution (5.11) elo. 8 100 mi volumetric tak (6.1) and d8Ao to tho mark wath ‘5.46. Tels butfericalclum chlorkde solution. Dissolve 6.05 g of Tris (5.14) and 735 mg of calcium chionde (5.8) in 900 mi of water in a 1! graduated measuring ‘offindar. Adjust the pH to 8.2 + 0,1 with hydrochloric acid (5.3) and dite tp 11 with water, 5.47 L-BAPA reagent, Prepare this reagent on tho day of use. Dissolve 60 mg of L!BAPA (5.13) in. 1 mi of DMSO (5.15) in & 100 mi ‘volumetrc ask (6.1) and cute lo the mark wtn Tris Buteticatcium chloride solution (5.16)ISSO 14902; 2001 6 Apparatus: Usual taboratory apparatus and, in particular, the folowing. 6.1 Volumetric Masks, of eapseity 100 mi 82 Cuvettes, wih optical path length 10 rim, 6.3. Refigerater, controled at a lemperalure of (4 3)°C. 6.4 pH:meter, wih an inaccuracy of 0,08 units 6.5 Test tubs mixer 6.6 Spectrometer, suitable for measuremonis at a wavelength of 410 nm. 6.7 Stopwaten. 5.8 Water bath, with circulation pump, capable of being maintained at (37 & 0,25) 5.9 Grinding apparatus, provided wit a 0,5 mm sieve. 6.10 Centrifuge, operating ata radial acceleration of approximately 1 500 ty 6.11 Centrifuge tubes, 7 Sampling I is important that the faboralory recaive a sampie which is truly representative and has not been damaged of changed during transport or storage Sampling if not part of the method specified In this Inlornational Standard. A eocammendad wompling method is given in 1S 6497 {5} 8 Preparation of test sample Using the grinding apparatus (68), grind a representative part of sample go thel heat prodvetion ip mlrimal, Mix he round sample thoroughly. 9 Procedure 8.1. Number of dotorminations It is required to chock whether tho repaatabilly limi (11.2) 's met, camry out two single determinations in accotdance with 0.2 and 0.5 under repealabily conditions, 92. Sample extraction Weigh 1.920.001 g of the prepared test sample (clause 8) in a 100m! conical tack and add $0 mi of socium hydroxide sehiten (5.2), Completely suspend the sample. Adjust he pH to 9.50, 1 with hyeroehlode acid (6 4 and 5.5} Rinse the elecvode with a itva water a possible. Close the conical fatk and store overnight (15, to.28 hn the rligerator (63), Place in the rafigerator tha quantty of water needed for making up the sample extractsISHSO 14902 ; 2001 "ter thu sarpia axicact to @ 100 ml volumetric task (6.1), chute to he mark wilh water from the refrigerator and ik Stora Um valumetig flask In tha refigeraler, Tha sample extract romaine stable for one day. After “ecmenation for 15 min, the samplo extract may be worked up further end dilvied as required. Diluitons depend athe expeeted TIA value of the sample aid are carried out with water at room amperaivre. 3 Dilution of sample extract he TIA value of the sample and prepare three diferent diutons of the sample extract on the basis of the scheme in Tabie A 1. so that it may be expectsd that as a resuit of tha TIA measurement (9.5) st least one * the Hee inhibition percentages obtained wil be within the range of 40 % ta 60%. Hinges cf the twee results is within Ihis range, the estimation should be adapted and the procedure repeated. 3.4 Measurement of trypsin activity af working solution f heck ie actwity of cach batch of trynsin (5.10). The difference between the abearbance of the working solution © ¥2}:an the absorbance of the blank (4, = t)) should be 0.380 £0,050. In thie is not the case, ehack the quality fo the trypsin (5.19), If necessary, take a fresh far of urypsin, Fipetie inte centrifuge tubes according to the following scheme: ‘BAPA reagem (8.17) Whe tb.4) Ale 366 (5.7) ‘ie the contents of the tubes with the test lube mixer (6.5) and place the lubea in tha water bath (6.8) for 10 min, Auld. wong coktion (5 12) ‘ihe (he contents of the tubes. with the {est tube mixer and place the centrifuge lubes back in the water bath, Afler 10 mn 25 of incubabon, add the following: Blank standard | Standard ont a Acmie 804 6.7) © 1 ‘tig the contents of the tubes with the test tube mixer ‘Cemntritage the tubes tor 10 min in the centrifuge (6.10) at a radial acesieration of sppronimately 1 500 gn. tsasute the absorbanon of the clear sohitions relative fo water In the spectromeler (8.8) at 410 am i exvette (6.2) 210mm These solutions comin stable for at loael 2 h,$5 Measurement of trypsin inhibitor activity Pipate into contifuge tubes according tp the fotowing scheme Prepare for each dilution of sample extract (9-3) 4 corresponding blank solution. Sample extract solutions and corresponding biank solutions shal be dealt wah simeitaneously in the procedure, induding centtuping. SAA reagent S17) ‘Dhue sare enact (93) vente (5.9) eae rid (57) swoula awa ola ‘Mix the contents of tno thos with the tast tube miner (6.5) and place the tubes in the wattar bath (6.8) for 10 min Ads the following | ~s | Standard | Blank sample | sample Topen warg wokon (5:12) rn + Pot ‘Mix the contents of the tubes with the lest tube mixer and place the centrifuge tubes back in the water bath (6.8). ‘Attar 10 min £ § ¢ incubation add the folowing: wi mi mil 1 0 1 ‘Acoue acid (5.7) Blank standard | Standard | Banksampie | Sample mi o ‘Mix the contents ofthe tubes with the test tube mixer Cenirfuge the tubes for 10 min in the centrifuge (6.10) at a radial acceleration of approximately 1 500 fy. Measure the absorbance of the clear tolutions relative fo water in tha spectrometer (6.6) at 410 nm in cuvette (6.2). mm “These solutions remain stable for at least 2h. 10 Calculation 10.1 Inhibition percentage of sample extract solutions Calculate the inhibition percentage of the sample extract solutions by the equation: a Use) Lda tend 4995, The)whore 1 isthe inhiotion percentage, in percent, 4 isthe absorbance ofthe sokiton with standard; for Ish absorbance ofthe blank with standard 4, Is the absorbance ofthe solution wih sample; ‘oy isthe absorbance ofthe blank wih sample 40.2 Trypsin Inhibitor activity ‘Calcuéste the trypsin inhibitor actly, expressed in sitigrams of inhibited trypsin par gram of samplo, by the equation hh TIA i the lrypsin init activi, it muligrams per gram; + in thu inhibition porcentage, in percent: rma) isha mass ofthe test sample, in grarns; ‘ay isthe mass of rypsin, in miligrams; {+ ish dition ofthe nample extract [100 mix 100 miyP, whore Mi the volume derived ftom Table A, inmilitres} flea conversion factor (2,8 x 10-4) based on the purty of trypsin (56%, see refs. 1] and [2)) and on the ution of ypsin according to 5.14 and 5.12 Round tha result to the nearest 0.4 mg/g ‘11 Precision 11.4 Interlaboratory tests ‘Details of interlaboratory tests on the precision of the mathod are given in annex B. The values derived from these tests may net bo applicable lo concentration ranges and matices other than thoes given, 11.2 Repeatability ‘The absolule difference between two in¢ependent single tes! results, oblained using the same method on Identical test material in he same laboratory by the sama operalor using the samo equipmant within a shon intarval of te, willin nol more than § % of cases excsed Une repeatablty lim (7) mentioned In or derived from Tabie 1,Table 1 — Repeatabiity limit (-) and reproductbility timit (#) 411.3 Reproducibility ‘The absolvte diflerence between two single test resuits, oblained using tha same method on igentical test material in different laboratories by different oparaiors using different equipment, wil in not more than 5% of cases exceed ‘the reproductilty mit (2) mentioned in or derived frem Table 1 12 Tost report The tost report shall specify: — ailinformation necessary for the complete idenification of the sample; — tha camping method uesd, # known; — the! ‘method used, with reference to this intemational Standart — all operating detalis not specified in this Inlamationat Standard, or regarded as optional, together with detalis of any inckients which may have iifluenced the last recutts; the test result obtained or, the repeatability has been checked, the two test results obtzined.Annex A (normative) Dilution scheme for sample extract Table A1 gives the dilution scheme to be folowed. Figures Ai and AZ show an example of a graphical reoresentation of the ditution scheme. Table A.1 — Dilution scheme ‘Theoretical dilution at diferent inhibition percentages Expected TA 11009