SIST EN ISO 8069:2007

SLOVENSKI STANDARD
SIST EN ISO 8069:2007
01-oktober-2007
0OHNRYSUDKX'RORþHYDQMHYVHEQRVWLPOHþQHNLVOLQHLQODNWDWRY,62
Dried milk - Determination of content of lactic acid and lactates (ISO 8069:2005)
Milchpulver - Bestimmung des Gehalts an Milchsäure und Lactaten (ISO 8069:2005)
Lait sec - Determination de la teneur en acide lactique et en lactates (ISO 8069:2005)
Ta slovenski standard je istoveten z: EN ISO 8069:2007
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
SIST EN ISO 8069:2007 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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EUROPEAN STANDARD
EN ISO 8069
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2007
ICS 67.100.10

English Version
Dried milk - Determination of content of lactic acid and lactates
(ISO 8069:2005)
Lait sec - Determination de la teneur en acide lactique et en Milchpulver - Bestimmung des Gehalts an Milchsäure und
lactates (ISO 8069:2005) Lactaten (ISO 8069:2005)
This European Standard was approved by CEN on 19 May 2007.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2007 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 8069:2007: E
worldwide for CEN national Members.

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EN ISO 8069:2007 (E)






Foreword



The text of ISO 8069:2005 has been prepared by Technical Committee ISO/TC 34 "Agricultural
food products” of the International Organization for Standardization (ISO) and has been taken
over as EN ISO 8069:2007 by Technical Committee CEN/TC 302 "Milk and milk products -
Methods of sampling and analysis", the secretariat of which is held by NEN.

This European Standard shall be given the status of a national standard, either by publication of
an identical text or by endorsement, at the latest by December 2007, and conflicting national
standards shall be withdrawn at the latest by December 2007.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United
Kingdom.


Endorsement notice

The text of ISO 8069:2005 has been approved by CEN as EN ISO 8069:2007 without any
modifications.

2

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INTERNATIONAL ISO
STANDARD 8069
IDF
69
Second edition
2005-09-15

Dried milk — Determination of content of
lactic acid and lactates
Lait sec — Determination de la teneur en acide lactique et en lactates




Reference numbers
ISO 8069:2005(E)
IDF 69:2005(E)
©
ISO and IDF 2005

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ISO 8069:2005(E)
IDF 69:2005(E)
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©  ISO and IDF 2005
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 either ISO or IDF at the respective
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Published in Switzerland

ii © ISO and IDF 2005 – All rights reserved

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ISO 8069:2005(E)
IDF 69:2005(E)
Contents Page
Foreword. iv
1 Scope . 1
2 Terms and definitions. 1
3 Principle. 1
4 Reagents. 1
5 Apparatus . 3
6 Sampling. 3
7 Preparation . 4
7.1 Preparation of test sample. 4
7.2 Test portion . 4
7.3 Blank test. 4
7.4 Preparation of solution and deproteination. 4
8 Procedure . 4
8.1 Test to check the activity of reagents. 4
8.2 Determination. 5
9 Calculation and expression of results. 6
9.1 Calculation. 6
9.2 Expression of results . 7
10 Precision. 7
10.1 Interlaboratory test . 7
10.2 Repeatability. 7
10.3 Reproducibility. 8
11 Test report . 8
Annex A (normative) Good laboratory practice (GLP) rules for the performance of enzymatic
analyses. 9
Bibliography . 13

© ISO and IDF 2005 – All rights reserved iii

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ISO 8069:2005(E)
IDF 69:2005(E)
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 2.
The main task of technical committees is to prepare International Standards. 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 document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 8069⎪IDF 69 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and
IDF.
This edition of ISO 8069⎪IDF 69 cancels and replaces ISO 8069:1986, which has been technically revised.

iv © ISO and IDF 2005 – All rights reserved

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ISO 8069:2005(E)
IDF 69:2005(E)
Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of
standard methods of analysis and sampling for milk and milk products.
Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the
National Committees for voting. Publication as an International Standard requires approval by at least 50 % of
the IDF National Committees casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. IDF shall not be held responsible for identifying any or all such patent rights.
ISO 8069⎪IDF 69 was prepared by the International Dairy Federation (IDF) and Technical Committee
ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF
and ISO.
All work was carried out by the Joint ISO-IDF Action Team on Lactose and lactate determination, of the
Standing Committee on Main components of milk, under the aegis of its project leader, Mr J Romero (US).
This edition of ISO 8069⎪IDF 69 cancels and replaces IDF 69:1987, which has been technically revised.
© ISO and IDF 2005 – All rights reserved v

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ISO 8069:2005(E)
INTERNATIONAL STANDARD
IDF 69:2005(E)

Dried milk — Determination of content of lactic acid and lactates
1 Scope
This International Standard specifies an enzymatic method for the determination of the lactic acid and lactates
content of all types of dried milk.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
lactic acid and lactates content
mass of substances determined by the procedure specified in this International Standard
NOTE It is expressed as milligrams of lactic acid per 100 g of non-fat solids.
3 Principle
A test portion of dried milk is dissolved in warm water. The fat and proteins are precipitated then filtered. The
filtrate is treated with the following enzymes and biochemical substances, added simultaneously, but acting in
sequence:
a) L-lactate dehydrogenase (L-LDH) and D-lactate dehydrogenase (D-LDH), in the presence of nicotinamide
adenine dinucleotide (NAD), to oxidize lactate to pyruvate and to convert NAD to its reduced form NADH;
b) glutamate pyruvate transaminase (GPT), in the presence of L-glutamate, to transform pyruvate into
L-alanine and to convert L-glutamate to α-ketoglutarate.
The amount of NADH produced is determined by spectrophotometric measurement at a wavelength of
340 nm, and is proportional to the lactic acid and lactates content.
4 Reagents
Use only reagents recognized analytical grade. The water used in the preparation of the enzyme solutions
shall be of at least doubly glass-distilled purity and the water used for other purposes shall be glass-distilled or
of at least equivalent purity.
4.1 Potassium hexacyanoferrate(II) solution, c(K [Fe(CN) ]⋅3H O) = 35,9 g/l.
4 6 2
Dissolve 35,9 g of potassium hexacyanoferrate(II) trihydrate in water. Dilute with water to 1 000 ml and mix.
4.2 Zinc sulfate solution, c(ZnSO⋅7H O) = 71,8 g/l.
4 2
Dissolve 71,8 g of zinc sulfate heptahydrate in water. Dilute with water to 1 000 ml and mix.
© ISO and IDF 2005 – All rights reserved 1

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ISO 8069:2005(E)
IDF 69:2005(E)
4.3 Sodium hydroxide solutions
4.3.1 Sodium hydroxide solution I, c(NaOH) = 10 mol/l.
Dissolve 400 g of sodium hydroxide in water. Dilute with water to 1 000 ml and mix.
4.3.2 Sodium hydroxide solution II, c(NaOH) = 0,1 mol/l.
Dissolve 4,0 g of sodium hydroxide in water. Dilute with water to 1 000 ml and mix.
4.4 Glycerol solution (C H O ), with a volume fraction of 50 % glycerol.
3 8 3
4.5 Ammonium sulfate solution, c[(NH ) SO ] = 3,2 mol/l.
4 2 4
Dissolve 422,84 g of ammonium sulfate in water. Dilute with water to 1 000 ml and mix.
4.6 Buffer solution, pH 10.
Dissolve 7,92 g of glycylglycine (C H N O ) and 1,47 g of L-glutamic acid (C H NO ) in about 80 ml of water.
4 8 2 3 5 9 4
Adjust the pH to 10,0 ± 0,1 at 20 °C with sodium hydroxide solution I (4.3.1). Dilute with water to 100 ml and
mix.
This solution may be kept for 3 months if stored in a refrigerator at between 0 °C and +5 °C.
4.7 Nicotinamide adenine dinucleotide solution (NAD).
Dissolve 350 mg of nicotinamide adenine dinucleotide (C H N O P ) in 10 ml of water.
21 27 7 14 2
This solution may be kept for 4 weeks if stored in a refrigerator at between 0 °C and +5 °C. When the solution
is being used, keep the vessel immersed in crushed ice.
4.8 L-Lactate dehydrogenase (L-LDH), from hog muscle suspension.
Dissolve 10 mg of L-lactate dehydrogenase suspension in 1 ml of glycerol solution (4.4). The pH of the
obtained suspension should be about 7. The specific activity of the L-lactate dehydrogenase
(L-LDH, EC 1.1.1.27) suspension shall be at least 5 500 units/ml at 25 °C. If not, prepare another L-LDH
suspension.
The L-LDH suspension may be kept for 12 months if stored in a refrigerator at between 0 °C and +5 °C. When
the suspension is being used, keep the vessel immersed in crushed ice.
4.9 D-Lactate dehydrogenase (D-LDH), from Lactobacillus leichmannii suspension.
Dissolve 5 mg of D-LDH suspension in 1 ml of ammonium sulfate solution (4.5). The pH of the obtained
suspension should be about 6. The specific activity of the D-lactate dehydrogenase (D-LDH, EC 1.1.1.28)
suspension shall be at least 1 500 units/ml at 25 °C. If not, prepare another D-LDH suspension.
The D-LDH suspension may be kept for 12 months if stored in a refrigerator at between 0 °C and +5 °C. When
the suspension is being used, keep the vessel immersed in crushed ice.
4.10 Glutamate pyruvate transaminase (GPT), from pig heart suspension.
Dissolve 20 mg of GPT suspension in 1,0 ml of ammonium sulfate solution (4.5). The pH of the obtained
suspension should be about 7. The specific activity of the glutamate pyruvate transaminase (GPT, EC 2.6.1.2)
suspension shall be at least 1 600 units/mI at 25 °C. If not, prepare another GPT suspension.
2 © ISO and IDF 2005 – All rights reserved

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ISO 8069:2005(E)
IDF 69:2005(E)
Add 1,0 ml of ammonium sulfate solution (4.5) to the 1 ml suspension with 20 mg of GPT and mix. Centrifuge
this 2,0 ml suspension containing 10 mg of GPT/ml at a radial acceleration of 4 000 g for 10 min. Transfer
1,0 ml of the clear supernatant liquid and discard the remaining solution and pellet.
The suspension may be kept for 12 months if stored in a refrigerator at between 0 °C and +5 °C. When the
suspension is being used, keep the vessel immersed in crushed ice.
4.11 Lithium L-lactate solution
Dissolve 50 mg of lithium L-lactate (C H O Li) in water. Dilute with water to 500 ml and mix.
3 5 3
4.12 Lithium D-lactate solution
Dissolve 50 mg of lithium D-lactate (C H O Li) in water. Dilute with water to 500 ml and mix.
3 5 3
5 Apparatus
Usual laboratory equipment and, in particular, the following.
5.1 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg.
5.2 Glass beaker, of capacity 50 ml.
5.3 Graduated cylinder, of capacity 50 ml.
5.4 One-mark volumetric flasks, of capacity 100 ml.
5.5 Pipettes, capable of delivering 0,02 ml, 0,05 ml, 0,2 ml, 1,0 ml and 2,0 ml.
5.6 Graduated pipettes, capable of delivering 5 ml and 10 ml, graduated in 0,1 ml divisions.
5.7 Glass filter funnel, of diameter about 7 cm.
5.8 Filter paper, medium fast grade, of diameter about 15 cm, free from lactic acid and lactates.
5.9 Glass rod.
5.10 Plastic paddles, capable of mixing the sample-enzyme mixture in the spectrometric cell.
5.11 Spectrophotometer, capable of measuring at 340 nm, equipped with cells of optical path length 1 cm.
TM1)
5.12 Parafilm .
6 Sampling
A representative sample should have been sent to the laboratory. It should not have 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 707⎪IDF 50.
Store the sample in such a way that deterioration and change in composition are prevented.

TM
1) Parafilm is an example of a product available commercially. This information is given for the convenience of users
of this International Standard and does not constitute an endorsement by ISO or IDF of this product.
© ISO and IDF 2005 – All rights reserved 3

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ISO 8069:2005(E)
IDF 69:2005(E)
7 Preparation
7.1 Preparation of test sample
Transfer the test sample to a container with cap
...