UGANDA STANDARD  
     US ISO 5510
 First Edition
    2009 -09-04
 
Reference numbe r
US ISO 5510:1984
 © UNBS 200 9 
  
Animal feeding stuffs — Determination of available lysine 
 US ISO 5510:1984  
 
© 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 5510:1984  
© 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 5510:1984,  Animal feeding stuffs — Determination of available lysine, is 
identical with and  has been reproduced from an International Standard ISO 5510:1984,  Animal 
feeding stuffs — Determination of available lysine, and has been adopted as a Uganda Standard . 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." International Standard 
INTERNATIONAL ORGANIZATION FOR STANDARDlZATIONWUlE~YHAPO~HAR OPTAHM3ALWl fl0 CTAH~APTbl3AL&lbl@ORGANlSATlON INTERNATIONALE DE NORMALlSATlON 
Animal feeding stuffs - Determination of available lysine 
Aliments des animaux - Dosage de la lysine disponible 
First edition - 1984~11-15 
iii UDC 636.087.1: 543.544: 547.466.46 Ref. No. IS0 5510-1984 (E) - 
Descriptors : animal feeding products, tests, determination of content, lysine. 
Price based on 5 pages 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, govern- 
mental and non-governmental, in liaison with ISO, also take part in the work. 
Draft International Standards adopted by the technical committees are circulated to 
the member bodies for approval before their acceptance aslnternational Standards by 
the IS0 Council. They are approved in accordance with IS0 procedures requiring at 
least 75 % approval by the member bodies voting. 
International Standard IS0 5510 was prepared by Technical Committee ISO/TC 34, 
Agricultural food products. 
0 International Organization for Standardization, 1984 
Printed in Switzerland INTERNATIONAL STANDARD IS0 55104984 (E) 
Animal feeding stuffs - Determination of available lysine 
0 Introduction 
Lysine in food products is generally considered to be 
assimilable when its terminal amino group (amino E) is not com- 
bined. This fraction of lysine can be determined because these 
groups combine with 2,4-dinitrofluorobenzene. 
1 Scope and field of application 
This International Standard specifies a method for the deter- 
mination of the available lysine in animal feeding stuffs contain- 
ing animal or vegetable proteins. 
Compared with biological determination, the method does, 
however, overestimate the amount of availabie lysine and care 
is necessary in interpreting the results. 
2 References 
IS0 5983, Animal feeding stuffs - Determination of nitrogen 
content and calculation of crude protein content. 
I SO 6497, Animal feeding stuffs - Sampling. 1 ) 
IS0 6498, Animal feeding stuffs - Preparation of test samples. 
3 Definition 
available lysine: The quantity of lysine corresponding to the 
difference between the total lysine and the non-available lysine 
determined under the conditions specified in this International 
Standard. 
It is expressed as a percentage by mass of the raw product. 
4 Principle 
Hydrolysis with hydrochloric acid of a ground test portion, 
followed by separation of the total Iysine by column 
chromatography and determination by spectrometry at 
570 nm. 
Reaction of a second ground test portion with an ethanolic 
solution of 2,4-dinitrofluorobenzene in an alkaline medium, 
1) At present at the stage of draft. purification, then hydrolysis with hydrochloric acid, separation 
of the non-available lysine by column chromatography, and 
determination by spectrometry at 570 nm. 
5 Reagents 
All the reagents shall be of recognized analytical grade. The 
water used shall be distilled water or water of at least equivalent 
purity. 
5.1 Diethyl ether, free from peroxides. 
5.2 Sodium bicarbonate, 80 g/l solution. 
5.3 2,4-Dinitrofluorobenrene (DNFB), ethanolic solution. 
Dissolve 0,15 ml of DNFB in 12 ml of 95 % ( Vl v) ethanol. 
Prepare this solution at the time of use. 
5.4 Hydrochloric acid, approximately 6 mol/l . 
Mix 1 volume of hydrochloric acid & = 1,19 g/ml) with 
1 volume of water. 
5.5 Sodium citrate, buffer solution, pH 2,2 approximately. 
Successively dissolve in water 
21 g of citric acid monohydrate; 
8 g of sodium hydroxide; 
16 ml of hydrochloric acid & = 1,19 g/ml); 
0,l ml of octanoic (caprylic) acid; 
20 ml of thiodiglycol; 
3 ml of a 30 % (Vl V) aqueous solution of polyoxyethylene 
dodecyl ether with approximately 23 molecules of oxy- 
ethylene? 
Dilute to 1 000 ml with water. 
2) A suitable commercially available product is BRIJ 35. This information is given for the convenience of the user of this International Standard and 
does not constitute an endorsement of this product by ISO. 
1 IS0 55104984 (El 
5.6 Sodium citrate, buffer solution, pH 5,28. 
Successively dissolve in water 6.1 Grinder, having the following characteristics: 
a) constructed of a material which does not absorb 
moisture; 
24,5 g of citric acid monohydrate; 
14 g of sodium hydroxide; 
6,8 ml of hydrochloric acid (eZO = 1,19 g/ml); b) easy to clean and having minimum dead space; 
c) permitting rapid and uniform grinding, without causing 
undue heating and preventing, as much as possible, contact 
with the outside air; 
0,l ml of octanoic (caprylic) acid; 
3 ml of a 30 % 1 V/v) aqueous solution of polyethoxyl 
dodecyl ether with approximately 23 molecules of oxy- 
ethylene. 1 ) d) capable of being regulated to give particles of the sizes 
specified in 7.1. 
Dilute to 1 000 ml with water. Adjust the pH to 5,28 + 0,02 
using hydrochloric acid (eZO = 1,19 g/ml) or 50 % (m/m) 
sodium hydroxide solution. 6.2 Flasks, of capacities 250 ml and 1 000 ml, flat- 
bottomed, short-necked with ground conical joints and reflux 
condensers to fit the flasks. 
5.7 Ninhydrin reagent. 6.3 Crucible, with a sintered glass plate of grade P 16 (pore 
size index 10 to 16 pm). 
57.1 Sodium propionate, buffer solution, pH 5,5 + 0,l. 
Dissolve 168 g of sodium hydroxide in about 400 ml of water. 
Cool, then add while shaking, 498 ml of propionic acid. Dilute 
to 1 000 ml with water. 6.4 Oil-bath, capable of being maintained at a temperature 
which will ensure refluxing (120 to 130 “C). 
6.5 Rotary evaporator. 
57.2 Preparation of reagent. 
Prepare the ninhydrin reagent in an atmosphere of nitrogen and 
in the dark. Into a 2 I flask, place 1 litre of peroxide-free 
ethylene glycol monomethyl ether, 1 litre of the sodium pro- 
pionate buffer solution (5.7.1) and 40 g of ninhydrin. Shake un- 
til completely dissolved, then add 1,33 g of tin(ll) chloride 
dihydrate (SnCl,.2H,O). Shake until completely dissolved. (See 
the note. 1 6.6 Apparatus for automatic analysis of amino acids, or 
if this is not available, manual chromatographic system 
comprising : 
This reagent is stable for 1 month if kept at 4 OC under light 
pressure in an atmosphere of nitrogen and in the dark. a) a chromatography column, of length about 250 mm 
and of internal diameter 6 mm, thermostatically maintained 
at 55 OC by means of a water jacket and circulation bath, 
and filled up to 200 mm with an 8 % cross-linked strongly 
acidic, cation exchange resin with sulfonic functional 
groups, of particle size 13 + 2 pm;2) 
b) small piston pump, producing a flow of 50 ml/h; 
c) fraction collector ; 
NOTE - If precipitation of SnCl2 occurs, replace the tin(H) chloride 
with 7,5 ml of 150 g/l titanium (Ill) chloride solution or by I,5 g of 
hydrindantin per litre of reagent. 
5.8 Lysine, standard solution, corresponding to 1 mmol of 
lysine base per litre. d) boiling water-bath; 
e) spectrometer, set at 570 nm, with cells of thickness 
10 mm. 
Dissolve l82,5 mg of lysine monohydrochloride in 100 ml of 
0,l mol/l hydrochloric acid. Take exactly 10 ml of this solution 
and dilute it to 100 ml with sodium citrate buffer solution of 
pH 2,2 (5.5). 6.7 Graduated pipettes, of capacities 1; 5; and 10 ml. 
6.8 Volumetric flasks, of capacities 10; 20; and 100 ml. 
6.9 Analytical balance. 
1 ml of this solution contains 1 pmol of lysine base. 
6 Apparatus 
Usual laboratory equipment,. and in particular: 7 Sampling 
See IS0 6497. 
1) A suitable commercially available product is BRIJ 35. This information is given for the convenience of the user of this International Standard and 
does not constitute an endorsement of this product by ISO. 
2) A suitable commercially available product is Aminex A5. This information is given for the convenience of the user of this International Standard 
and does not constitute an endorsement of this product by ISO. 
2 IS0 55104984 (E) 
8 Procedure 
8.1 Preparation of the test sample 
Grind 5 to 10 g of the laboratory sample to obtain particles 
which will pass completely through a sieve of aperture size 
315 pm. 
NOTE - The aperture size specified is smaller than that recommended 
in IS0 6498 in order to ensure maximum contact with the DNFB. 
8.2 Test portions 
Weigh, to the nearest 1 mg, and place respectively in the two 
flasks (6.2), two test portions each of which corresponds to 
approximately 100 mg of crude protein. (See IS0 5983 for the 
determination of the protein content.) 
8.3 Total Iysine 
8.3.1 Hydrolysis with hydrochloric acid 
Into the 1 000 ml flask (see 8.2), place 500 ml of the 6 mol/l 
hydrochloric acid (5.4). Fit a reflux condenser and place the 
flask in the oil-bath (6.4), previously heated to 120 to 130 OC. 
After boiling gently for 24 h, cool the flask and filter the con- 
tents through the crucible (6.3). Evaporate the filtrate at a tem- 
perature not exceeding 40 OC, using the rotary evaporator 
(6.5). Add water to the residue thus obtained and evaporate. 
Repeat this operation until most of the hydrochloric acid has 
been removed; in general, four rinses with 30 ml of water are -- 
sufficient. 
Dissolve the residue in sodium citrate buffer solution of pH 2,2 
(5.5) and transfer quantitatively to a 100 ml one-mark 
volumetric flask (6.8). Make up to the mark with the sodium 
citrate buffer solution of pH 2,2 (5.5). Filter through a folded 
filter paper. 
8.3.2 Final preparation of the column and adsorption of 
the hydrolysate 
Connect the pump of the apparatus (6.6) to a reservoir contain- 
ing sodium citrate buffer solution of pH 528 (5.6); then adjust 
it to obtain a flow of 50 ml/h. Connect the pump to the resin 
column, previously heated to 55 OC, and allow the buffer solu- 
tion (5.6) to pass through the column for 20 min in order to 
establish equilibrium. Disconnect the pump. Remove most of 
the liquid above the resin, taking care that the surface of the 
resin does not become dry. 
By means of a pipette (6.7), take 0,5 ml of hydrolysate (or 1 ml 
if using the manual system), place it on the column, then allow 
it to pass through the resin with the aid of a slight nitrogen 
pressure. Rinse the walls of the column with 0,5 ml of sodium 
citrate buffer solution of pH 5,28 (5.6) and pass through the 
resin. Fill the column to the top with sodium citrate buffer solu- 
tion (5.6) and connect it to the pump. 
8.3.3 Determination 
8.3.3. I Automatic system 
Set the analyser in operation. Calibrate the apparatus, pro- 
ceeding as described in 8.3.2 using 0,25 prnol of lysine [0,25 ml of the lysine standard solution (5.8)l or the amount specified in 
the manufacturer’s instructions. 
8.3.3.2 Manual system 
8.3.3.2.1 Localization of lysine 
The fraction of eluate containing the lysine shall be checked by 
using a lysine so!ution of known concentration, for example the 
1 ~mol/ml solution (5.8). For this purpose, discard the first 25 
to 30 ml of eluate and then collect fractions of 1 ml in test- 
tubes until the 50th fraction is obtained, reckoned from the 
beginning of the elution. Add to each fraction, between the 
30th and the 50th fractions, 1 ml of the ninhydrin reagent (5.7). 
Mix, transfer to the boiling water-bath and leave for 15 min. 
Cool. Dilute by adding 10 ml of sodium citrate buffer solution 
of pH 5,28 (5.6). Mix and measure the absorbance at 570 nm, 
by means of the spectrometer, using sodium citrate buffer solu- 
tion of pH 5,28 (5.6) as the reference liquid. 
NOTE - For information only, under the conditions described, lysine 
generally elutes between the 39th and 45th fractions. 
8.3.3.2.2 Determination 
If the lysine is eluted between the 39th and 45th fractions 
discard the first 38 ml of eluate, collect and combine the frac- 
tions (Nos 39 to 45) corresponding to the lysine peak and 
evaporate using the rotary evaporator (6.5). 
NOTE - After recovering the fractions containing the lysine, allow 
about 200 ml of buffer solution to pass through the column in order to 
remove any undesirable components which may remain. 
Dissolve the residue in 5 ml of the sodium citrate buffer solu- 
tion of pH 5,28 (5.6) and add 5 ml of the ninhydrin reagent 
(5.7). Mix, transfer to the boiling water-bath and leave for 
15 min. Cool. Dilute the mixture with the sodium citrate buffer 
solution of pH 5,28 (5.6) so that the lysine concentration of the 
test solution is approximately O,O2 (Imol/ml. (Let V, be the 
volume of test solution thus obtained.) 
Measure the absorbance at 570 nm by means of the spec- 
trometer, using as the reference liquid a”i’i’iixtwre-of one part by 
volume sodium citrate buffer solution (5.6) and%e- part by 
volume ninhydrin reagent (5.7), which has been placed for 
15 min in the boiling water-bath and diluted to the volume V, 
after cooling. 
- ‘-.A< v-- _, .--- c- 
83.3.2.3 Calibration of the spectrometer ?. -..- 
Take exactly 5 ml of the standard lysine solution (5.8). Add 
5 ml of the ninhydrin reagent (5.7). Mix, transfer to the boiling 
water-bath and leave for 15 min. Cool and dilute to 100 ml with 
sodium citrate buffer solution of pH 5,28 (5.6). 
Measure the absorbance, by means of the spectrometer, under 
the same conditions as in 8.3.3.2.2. 
8.4 Non-available lysine 
8.4.1 Dinitrophenylation reaction 
By means of a pipette (6.7), transfer 8 ml of the sodium bicar- 
bonate solution (5.2) to the 250 ml flask (see 8.2). Leave for IS0 5510-1984 (E) 
about 10 min, stirring from time to time. Then add the DNFB 
solution (5.31, stopper the flasks, agitate, and ensuring that 
there are no particles adhering to the walls of the flask, leave 
the mixture overnight at ambient temperature and in darkness. V. is the volu me, in millilitres 
to the column ( wn erally, V. = of hydrolysate transferred 
‘0,5 ml). 
9.1.1.2 Determination using a manual system 
The total 
equal to lysine, Wlf expressed as a percentage by mass, is 8.4.2 Purification 
Evaporate to dryness at a temperature below 40 OC, using the 
rotary evaporator (6.5). Add 75 ml of the diethyl ether (5.1) to 
the flask, stir and allow to separate. Pour off most of the diethyl 
ether, taking care to avoid entraining solid particles with it. 
Repeat these operations twice more, using each time 50 ml of 
the diethyl ether. Evaporate the last traces of diethyl ether by 
heating on the rotary evaporator. v, x 0,073 A, 
X- 
ml x Fl A0 
A0 is the absorbance of the standard lysine solution deter- 
mined in 8.3.3.2.3; 
8.4.3 Hydrolysis with hydrochloric acid 
A, is the absorbance determined in 8.3.3.2.2; 
Transfer to the flask 150 ml of the hydrochloric acid (5.4) and 
proceed as described in 8.3.1 but transfer quantitatively the 
residue, dissolved in sodium citrate buffer solution of pH 2,2 
(5.5) to a 20 ml volumetric flask (6.8) (or 10 ml if a manual 
system is being used). V. is the vol ume, in millilitres, of hydrolysate 
to the column (generally, V. = 1 ml); transferred 
h is the volume, in millilitres, of the test solution ; 
ml is the 
first flask. mass, in grams, of the test portion 8.4.4 Deposition of the hydrolysate and determination . 
Proceed as described in 8.3.2 and 8.3.3. 
9.1.2 Non-available lysine 
In the case of the manual system, the final reading on the spec- 
trometer may be made by diluting the volume of the lysine plus 
reagent mixture to 20 ml with sodium citrate buffer solution of 
pH 5,28 (5.6) (Let V2 be the volume of test solution thus 
obtained.) 9.1.2.1 Determination using an automatic analyser 
The non-available lysine, w2, expressed as a percentage by 
mass, is equal to 
0,073 s* 
m2 x b “s, 85 . Number of determinations 
Carry out two determinations on the 
two d ifferent pairs of test portions. same test sample using 
So is the area of the peak corresponding to 0,25 pmol of 
lysine ; 
S2 is the area of the peak corresponding to the non- 
available lysine, determined in 8.4.4; 9 Expression of results 
9.1 Method of calculation and formulae 
m2 is the mass, 
second flask; in grams, of the test portion placed in the 
9.1.1 Total lysine 
V. is the vol 
to the column U me, in millilitres, of hydrolysate tra 
generally, V. = 0,5 ml). nsferred 9. I. I. 1 Determination using an automatic analyser 
The total lysine, wl, expressed as a percentage by mass, is 
equal to 
0,365 S, 
----------Xx 
mlxI/o So 9.1.2.2 Determination using a manual system 
The non-available 
mass, is equal to lysine, w2, expressed as a percentage bY 
where V2 x 0,007 3 A2 
X- 
m2 x I/o A0 So is 
lysine; the area of the peak corresponding to 0,25 pmol of 
S, is the area of the peak corresponding 
determined in 8.3 .3.1; to the total lysine 
A0 is the absorbance of the standard lysine solution deter- 
mined in 8.3.3.2.3; 
ml is the mass, in grams, of the test portion placed in the 
first flask; is the absorbance determined in 8.4.4; IS0 5510-1984 E) 
V. is the volume, in millilitres, of hydrolysate transferred NOTE - The available lysine, expressed as a percentage by mass of 
to the column (generally, V. = 1 ml); total lysine is given by the formula 
V2 is the volume, in millilitres, of the test solution (gener- 
ally, v2 = 20 ml); iwl - w$ - x 100 
w1 
m2 is the mass, in grams, of the test portion placed in the 9.2 Repeatability 
second flask. 
9.1.3 Available lysine The difference between the results of two determinations 
carried out simultaneously or in rapid succession by the 
same analyst shall not exceed 10 % of the mean value of the 
results obtained. 
The available lysine, w3, expressed as a percentage by mass, is 
equal to 10 Test report 
Wl --W2 The test report shall show the method used and the results ob- 
tained. It shall also mention all operating details not specified in 
where this International Standard, or regarded as optional, together 
with details of any incidents likely to have influenced the 
results. 
w1 is the total lysine (see 9.1 .I) ; 
is the non-available lysine (see 9.1.2). The test report shall include all the information 
the complete identification of the sample. necessary for This page intentionally left blank