UGANDA STANDARD  
      US ISO 6655
First Edition
   2009 -04-09
 
Reference numbe r 
   US ISO 6655:1997
 © UNBS 200 9 
  
Animal feeding stuffs — Determination of soluble nitrogen 
content after treatment with pepsin in dilute hydrochloric acid 
 US ISO 6655:1997  
 
© 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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    US ISO 6655:1997  
© 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 6655:1997,  Animal feeding stuffs — Determination of soluble nitrogen content
after treatment with pepsin in dilute hydrochloric acid, is identical with and  has been reproduced from an
International Standard ISO 6655:1997,  Animal feeding stuffs — Determination of soluble nitrogen content
after treatment with pepsin in dilute hydrochloric acid, and has been adopted as a Uganda Standard.
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." INTERNATIONAL 
STANDARD IS0 
6655 
First edition 
1997-08-01 
Animal feeding stuffs - Determination of 
soluble nitrogen content after treatment 
with pepsin in dilute hydrochloric acid 
Aliments des animaux - Dktermina tion de la teneur en azote soluble apr& 
traitement avec de la pepsine dans I’acide chlorhydrique dilu& 
Reference number 
IS0 6655: 1997(E) IS0 6655:1997(E) 
Foreword 
IS0 (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS0 
member bodies). The work of preparing International Standards is normally carried out through IS0 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. IS0 collaborates closely with the International Electrotechnical 
Commission (IEC) on all matters of electrotechnical standardization. 
Draft Intern ational Standards adopted by the technical co mmittees a ,re circul ated to the member bodies for votin 
Publication as an I nternational Standard requi res approval by at least 75 % of the me ‘mber bodies casting a vote. g . 
International Standard IS0 6655 was prepared by Technical Committee ISOmC 34, Agricultural food products, 
Subcommittee SC IO, Animal feeding stuffs. 
Annex A forms an integral part of this International Standard. 
Annexes B and C are for information only. 
0 IS0 1997 
All rights reserved. Unless otherwise specified, no part 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 the publisher. 
International Organization for Standardization 
Case postale 56 l CH-1211 Geneve 20 l Switzerland 
Internet central @ iso.ch 
x.400 c=ch; a=400net; p=iso; o=isocs; s=central 
Printed in Switzerland 
ii INTERNATIONAL STANDARD @ iso IS0 6655:1997(E) 
Animal feeding stuffs - Determination of soluble nitrogen content after 
treatment with pepsin in dilute hydrochloric acid 
1 scope 
This International Standard specifies a method for the determination of the soluble nitrogen content of 
animal feeding stuffs after treatment with pepsin in dilute hydrochloric acid. 
This method does not distinguish between protein nitrogen and non-protein nitrogen. 
NOTES 
1 The values obtained on using this method have no direct connection with digestibility in vivo. 
2 %d non-protein nitrogen is to be excluded from the test result, its content should be determined using an appropriate 
method. 
2 Normative references 
The following standards contain provisions which, through reference in this text, constitute provisions of 
this International Standard. At the time of publication, the editions indicated were valid. All standards are 
subject to revision, and parties to agreements based on this International Standard are encouraged to 
investigate the possibility of applying the most recent editions of the standards indicated below. Members 
of IEC and IS0 maintain registers of currently valid International Standards. 
IS0 5983: 1997’, Animal feeding stuffs - Determination of nitrogen content and calculation of crude protein 
content- Kjeldahl method. 
IS0 6498:1983, Animal feeding stuffs - Preparation of test samples. 
3 Principle 
Incubation of the sample for 48 h at 40 OC in a solution of pepsin in dilute hydrochloric acid. 
Filtration of the suspension and determination of the nitrogen content of either the filtrate or the residue 
remaining in the filter in accordance with the Kjeldahl method specified in IS0 5983. In the latter case, 
determination of the nitrogen content of the sample as well, in accordance with IS0 5983. 
4 Reagents 
Use only reagents of recognized analytical grade, and distilled or demineralized water or water of at least 
equivalent purity. 
The reagents (except the standard materials) shall be practically free from nitrogenous compounds. 
’ To be published. (Revision of IS0 5983:1979) IS0 6655: 1997(E) @ IS0 
4.1 Dilute hydrochloric acid, c(HCI) = 0,075 mol/l. 
4.2 Pepsin, having an activity of 2,0 units per milligram in accordance with the definition given in 
annex A. Check the pepsin activity in accordance with the method specified in annex A. 
4.3 Pepsin solution in hydrochloric acid, with a pepsin activity of about 400 units per litre. 
IDissolve 0,2 g k 0,001 g of pepsin (4.2) in 1 I of dilute hydrochloric acid (4.1). Prepare this solution 
immediately before use. 
If the activity of the pepsin deviates from 2,0 units per mil 
solution with a pepsin activity of 400 units per litre. 
44 . Hydrochloric acid, c(HCI) = 7,5 mol/l (~20 = 1,125 g/m igram, adjust the mass of pepsin to obtain a 
5 Apparatus 
Usual laboratory apparatus and, in particular, the following. 
51 . Water bath or incubator, capable of being maintained at 40 “C k 1 OC. 
52 . Kjeldahl digestion flasks, of suitable capacity. 
53 . Filter paper, fast filtration rate, acid-resistant. 
54 . Distillation and titration equipment 
6 Sampling 
Sampling is not part of the method specified in this International Standard. A recommended sampling 
method is given in IS0 6497 [3]. 
It is important that 
or changed during the laboratory receive a sample which is truly representative and has not 
transport or storage. damaged 
7 Preparation of test sample 
Prepare the test sample in accordance with IS0 6498. 
If the fat content of the test sample exceeds IO % (m/m), extract the fat in accordance with IS0 6498, and 
take the content of extracted fat into account in the calculation (clause 9). 
8 Procedure 
81 . Test portion 
Weigh, to the nearest 0,001 g, about 2 g of the prepared test sample. 
82 . Incubation 
Place the test portion in a 500 ml volumetric flask and add 450 ml of the pepsin solution (4.3) previously 
heated to 40 OC. Shake so as to avoid agglomeration. Check that the pH of the suspension is lower than 1,7. 
Place the flask in the water bath or incubator (5.1) set at 40 OC and leave it there for 48 07. Shake after 8 h, 
24 h and 32 h. 
2 @ IS0 IS0 6655:1997(E) 
After 48 h, add 15 ml of hydrochloric acid (4.4) and cool to 20 ‘Cm Dilute to the mark with water, shake and 
filter through filter paper (5.3). Proceed in accordance with 8.3 or 8.4. 
83 . Determination of the nitrogen content of the filtrate 
8.3.1 Digestion of organic matter 
Take 250 ml of the filtrate (8.2) and transfer it into a Kjeldahl digestion flask (5.2). Add the reagents 
necessary for digestion as specified in IS0 5983:1997, 8.2.1. Mix and boil. 
NOTE - It may be advisable to add an antifoaming agent. 
Keep the solution boiling vigorously until the water has almost completely evaporated. Eliminate the last 
traces of water with care, reducing the rate of heating. After the liquid has become clear, continue heating 
for 1 h, then leave to cool. 
8.3.2 Distillation and titration 
Proceed as specified in IS0 5983:1997,8.2.2 and 8.2.3. 
8.3.3 Blank test 
Carry out 
clause 9. 
84 . 
8.4.1 a blank test using the same procedure but omitting the test portion. Proceed in accordance with 
Determination of the nitrogen content of the residue and of the test sample 
Digestion of organic matter in the residue 
Wash the filter paper and residue (8.2) with warm water until they are free from acid. Transfer the filter 
paper with residue to a Kjeldahl digestion flask (5.2). Add the reagents necessary for digestion as specified 
in IS0 5983:1997, 8.2.1. Mix and boil. 
NOTE - It may be advisable to add an antifoaming agent. 
Remove the water by boiling vigorously initially and next by reducing the rate of heating to eliminate the 
last traces of water. After the liquid has become clear, continue heating for 1 h, then leave to cool. 
8.4.2 Distillation and titration 
Proceed as specified in IS0 5983:1997, 8.2.2 and 8.2.3. 
8.4.3 Determination of the nitrogen content of the test sample 
Determine the nitrogen content of the prepared test sample (see clause 7) in accordance with IS0 5983. 
8.4.4 Blank test 
Carry out a blank test using the same procedure but omitting the test portion. 
9 Expression of results 
91 . Calculation of the soluble nitrogen content obtained using the procedure specified in 8.3 
Provided that the quantities of sulfuric acid used to collect the ammonia for the determination and for the 
blank test are equal (see IS0 5983), calculate the soluble nitrogen content of the test sample by the equation 
WI = 2(v~-V,)xcxM 
m IS0 6655: 1997(E) @ IS0 
where 
w1 is the soluble nitrogen content, in grams per kilogram, of the test sample obtained by the 
procedure specified in 8.3; 
C is the concentration, in moles per litre, of the sodium hydroxide solution (see IS0 5983:1997, 49.1) 
used for the titrations; 
m is the mass, in grams, of the test portion; 
M is the molar mass, in grams per mole, of nitrogen (M = 14 g/mol); 
"0 is the volume, in millilitres, of the sodium hydroxide solution (see IS0 5983:1997, 4.9.1) used for 
the blank test; 
“I is the volume, in millilitres, of the sodium hydroxide solution (see IS0 5983:1997, 4.9.1) used for 
the determination (8.3.2). 
Report the result to the nearest 0,l g/kg. 
9.2 Calculation of the soluble nitrogen content obtained using the procedure specified in 8.4 
Provided that the quantities of su Ifu ri c acid used to toll ect the amm onia for the determinatio n and for the 
blank test are equal (see IS0 59 83), ca Iculate the solu ble nitroge n con tent of the test sample by the equation 
=w PO-“l)xcxM 
w2 N- m 
where 
w2 is the soluble nitrogen content, in grams per kilogram, of the test sample obtained by the 
procedure specified in 8.4; 
C 
m 
M is the concentration, in moles per litre, of the sodium hydroxide solution (see IS0 5983:1997, 4.9.1 
used for the titrations; 
is the mass, in grams, of the test portion; 
is the molar mass, in grams per mole, of nitrogen (M = 14 g/mol); 
"0 is the volume, in millilitres, of the sodium hydroxide solution (see IS0 5983:1997, 4.9.1) used fo 
the blank test; 
“1 
wN is the volume, in millilitres, of the sodium hydroxide solution (see IS0 5983:1997, 4.9.1) used for 
the determination (8.4.2); 
is the nitrogen content, in grams per kilogram, of the test sample determined in 8.4.3. 
Report the result to the nearest 0,l g/kg. 
93 . Calculation of soluble crude protein content 
If it is desired to express the result as soluble crude protein, multiply the soluble nitrogen content 
determined by the factor 6,25. 
10 Precision 
10.1 Interlaboratory test 
Details of an interlaboratory test on the precision of the method are given in annex B. The values derived 
from this interlaboratory test may not be applicable to concentration ranges and matrices other than those 
10.2 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, will in not more than 5 % of cases exceed the value of rshown in table 1. 
4 @ IS0 IS0 6655:1997( E) 
10.3 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 value of W shown in table I. 
Table 1 - Repeatability limit (I-) and reproducibility limit (R) 
Maize gluten feed 
Coconut meal 
Grass silage 
Bone meal 
Feather meal 
I) Based on dry matter. 512,0 89 I 63,l 
574,3 22,l 166,3 
II Test report 
The test report shall specify: 
all information necessary for the complete identification of the sample; 
the method with which sampling was carried out, if known; 
the method used; 
the test result(s) obtained, expressed as soluble nitrogen or soluble crude protein 
case the conversion factor used (i.e. 6,25); 
if the repeatability has been checked, the final quoted result obtained; and, in the latter 
all operating details not specified in this International Standard, or regarded as optional, together 
with details of any incidents occurred when performing the method, which may have influenced 
the test result(s). IS0 6655:1997(E) 
Annex A 
(normative) 
Determination of pepsin activity 
Al . Scope 
This annex specifies a method for the determination of the activity of the pepsin used in the determination 
of soluble nitrogen content after treatment with pepsin in dilute hydrochloric acid. 
2 Definition 
For the purposes of this annex, the following definition applies. 
A.2.1 unit of pepsin activity: That quantity of pepsin which liberates per minute, under the conditions 
specifiedl a quantity of hydroxyaryl groups whose coloration by Folin-Ciocalteu’s reagent has an 
absorbance equivalent to that of 1 pmol of tyrosine under the same conditions. 
A3 . Principle 
Treatment of h aemoglobin with 
protein fractio n by trichloroacetic pepsi n in dilute hydrochloric acid. Precipitation of the non-hydrolysed 
acid. 
Filtration and addition of sodium hydroxide and Folin-Ciocalteu’s reagent. Measurement of the absorbance 
of this solution at a wavelength of 750 nm and determination of the corresponding quantity of tyrosine from 
a calibration curve. 
A.4 Reagents 
Use only reagents of recognized analytical grade, and distilled or demineralized water or water of at least 
equivalent purity. 
A.4.1 Hydrochloric acid, c(HCI) = 0,2 mol/l. 
A.4.2 Hydrochloric acid, c(HCI) = 0,06 mol/l. 
A.4.3 Hydrochloric acid, c(HCI) = 0,025 mol/l. 
A.4.4 Trichloroacetic acid solution, p(CCI,CO,H) = 50 g/l. 
A.4.5 Sodium hydroxide solution, c(NaOH) = 0,5 mol/l. 
A.4.6 Folin-Ciocalteu’s reagent. 
Place 100 g of sodium tungstate dihydrate (Na,W0,.2H,O), 25 g of sodium molybdate dihydrate 
(Na,Mo0,.2H,O) and 700 ml of water in a 2 I round-bottomed flask with a ground glass neck. Add 50 ml of 
phosphoric acid [p(H,PO,) = I,71 g/ml] and 100 ml of concentrated hydrochloric acid [p(HCI) = I,19 g/ml]. Fit 
a reflux condenser to the flask, bring the solution to the boil and keep gently boiling for IO h. Allow to cool, 
detach the reflux condenser, add 175 g of lithium sulfate dihydrate (Li,S0,.2H,O), 50 ml of water and 1 ml of 
bromine. Boil for 15 min to eliminate the excess bromine. 
Allow to cool then decant the solution into a 1 I volumetric flask. Dilute to the mark with water, mix and 
filter. No greenish colouring shall remain. 
5efore use, dilute I volume of this reagent with 2 volumes of water. @ IS0 IS0 6655:1997(E) 
A.47 Haemoglobin solution. 
Weigh a quantity of haemoglobin-protein substrate according to Anson’s method (about 2 g) corresponding 
to 354 mg of nitrogen and place it in a 200 ml flask with a ground glass neck and a reference line at 100 ml. 
NOTE - If necessary, determine the nitrogen content of the substrate using a semi-micro Kjeldahl method. The 
theoretical nitrogen content is 17,7 % (m/m). 
Add a few millilitres of dilute hydrochloric acid (A.4.2), connect the flask to a vacuum pump and shake until 
the haemoglobin has completely dissolved. Release the vacuum and add, while shaking continuously, dilute 
hydrochloric acid (A.4.2) to make up to approximately 100 ml. 
Prepare this solution immediately before use. 
AA.8 Tyrosine standard solution, c(C,H,,NO,) = 0,2 mmol/l. 
To prepare a stock solution, dissolve in a 1 I volumetric flask 181,2 mg of tyrosine in dilute hydrochloric acid 
(A.4.1) and dilute to the mark with the same. 
Using a pipette, transfer 20 ml of the stock solution to a 100 ml volumetric flask. Add a quantity of dilute 
hydrochloric acid (A.4.1) and dilute to the mark with the same. 
The tyrosine concentration of this standard solution is 0,2 pmol/ml (= 0,2 mmol/l). 
A5 . Apparatus 
Usual laboratory apparatus and, in particular, the following. 
A.5.1 Water bath, capable of being maintained at 25 OC k 1 OC. 
A.52 Spectrometer, capable of carrying out measurements at a wavelength of 750 nm. 
A.53 Precision chronometer, capable of being read to the nearest 1 s. 
A.5.4 pH-meter, capable of being read to the nearest 0,l pH unit. 
A.5.5 Glass rod, thicker at one end than at the other. 
A.6 Procedure 
A.6.1 Preparation of the solution 
Dissolve 150 mg of pepsin (or the quantity necessary to obtain an absorbance value of 0,35 i= 0,035) in 
100 ml of dilute hydrochloric acid (A.4.2). 
Using a pipette, transfer 2 ml of this solution to a 50 ml volumetric flask and dilute to the mark with dilute 
hydrochloric acid (A.4.3). The pH of this solution shall be I,6 * 0,l. 
Immerse the flask in the water bath (A.5.1) set at 25 OC . 
A.6.2 Hydrolysis 
Using a pipette, transfer 5,0 ml of the haemoglobin solution (8.4.7) to a test tube and heat to a temperature 
of 25 OC k 1 OC in the water bath (A.5.1). Add I,0 ml of the pepsin solution obtained in A.6.1 and mix using a 
glass rod (A.5.5) with about 10 backwards and forwards movements. Keep the test tube in the water bath at 
25 OC k 1 OC for 10 min of 1 s, timed from the addition of the pepsin solution. The duration and temperature 
7 IS0 6655: 1997(E) @ IS8 
shall be precisely observed. Add IO,0 ml of the trichloroacetic acid solution (A.4.4), which has previously 
been heated to a temperature of 25 OC t 1 “C, mix and filter through a dry filter paper. 
A.6.3 Colour development and measurement of the absorbance 
Using a pipette, transfer 5,0 ml of the filtrate to a 50 ml conical flask, add IO,0 ml of the sodium hydroxide 
solution (A-4.5) and, shaking continuously, 3,0 ml of the diluted Folin-Ciocalteu”s reagent (A-4.6). After 5 min 
PO 10 min, determine the absorbance of the solution against water at a wavelength of 750 nm using the 
spectrometer (A-5.2) and a cell of 1 cm optical path length. 
A-6.4 Wan k test 
For each determination, carry out a blank test as follows. 
Using a pipette, transfer 5,0 ml of the haemoglobin solution (A-4.7) to a test tube. Heat to a temperature of 
25 “C * 1 “C in the water bath (A-5.1), add IO,0 ml of the trichloroacetic acid solution (A-4.4), which has 
previously been heated to a temperature of 25 OC - * 1 OC, mix and add I,0 ml of the pepsin solution obtained 
in A-6.1. Mix using a glass rod and keep the test tube in the water bath (A-5.1) at 25 OC t 1 OC for 
10 min k 1 s. Mix and filter through a dry filter paper. Follow the procedure specified in A-6.3. 
A.6.5 Calibration curve 
In a series of six 50 ml conical flasks, place 0 ml (zero member), I,0 ml, 2,0 ml, 3,0 ml, 4,0 ml and 5,0 ml of 
the tyrosine standard solution (A-4.8), corresponding respectively to 0 pmol, 0,2 pmol, 0,4 pmol, 0,6 pmol, 
0,8 pmol and I,0 pmol of tyrosine. Add to each flask, as appropriate, a sufficient quantity of dilute 
hydrochloric acid (A-4.1) to give a total volume of 5,0 ml in the flask. 
To each flask, add IO,0 ml of the sodium hydroxide solution (A-4.5) and, shaking continuously, 3,0 ml of the 
diluted Folin-Ciocalteu’s reagent (A-4.6). Measure the absorbance of each solution as indicated in A-6.3. 
Plot the calibration curve showing the absorbances against the quantities of tyrosine in micromoles. 
A7 . 
From the cali brat ion curve, 
absor bance of the coloured s Expression of results 
determ 
Iolution, ine the quantity of tyrosi n 
corrected for the blan k test. e, in micromoles, corresponding to the 
Calculate the pepsin activity at 25 “C k 1 “C, expressed in micromoles of tyrosine per milligram and per 
minute, by the equation 
3,2 n a= - 
mxt 
where 
a is the pepsin activity at 25 OC k 1 “C, in micromoles per milligram per minute; 
m is the mass, in milligrams, of pepsin used in A-6.1; 
n is the quantity, in micromoles, of tyrosine read from the calibration curve; 
t is the reaction time, in minutes (t= 10 min). 
NOTE 6 - Two units of pepsin activity per milligram obtained by this method correspond to 3,64 milliunits Anson per 
milligram (micromoles of tyrosine per milligram per minute at 35,5 “C) or 36 400 commercial units per gram 
(micromoles of tyrosine per gram in 10 min at 35,5 OC). ISO6655:1997( E) 
Annex B 
(informative) 
Results of interlaboratory test 
The precision of the method was established by an interlaboratory test, carried out at the international level 
by ISOnC 34/SC 10 in accordance with IS0 5725-2 [2]. In this test 19 laboratories participated, each of which 
carried out three determinations on each of six samples. 
1) Based on dry matter. 
9 IS0 6655:1997(E) @ IS0 
Annex @ 
(informative) 
Bibliography 
[I 1 IS0 5725-1:1994, Accuracy (trueness and precision8 of measurement methods and results.- Part 7: 
General principles and definitions. 
[2] IS0 5725-2:1994, A ccuracy (trueness and precision) of measurement methods and results.-Part 2: A 
basic method for the determination of repeatability and reproducibility of a standard measurement method. 
[3] IS0 6497:- 2 I Animal feeding stuffs - Sampling. 
2 To be published. 
10 This page intentionally left blank This page intentionally left blank This page intentionally left blank ISO6655:1997(E) 
ICS 65.120 
Descriptors: agricultural products, food products, animal feeding products, chemical analysis, determination of content, nitrogen. 
Price based on IO pages