Dried milk - Determination of content of lactic acid and lactates (ISO 8069:2005)

ISO 8069|IDF 69:2005 specifies an enzymatic method for the determination of the lactic acid and lactates content of all types of dried milk.

Milchpulver - Bestimmung des Gehalts an Milchsäure und Lactaten (ISO 8069:2005)

Diese Internationale Norm legt ein enzymatisches Verfahren für die Bestimmung des Gehalts an Milchsäure und Lactaten für alle Arten von Milchpulver fest.

Lait sec - Determination de la teneur en acide lactique et en lactates (ISO 8069:2005)

L'ISO 8069|FIL 69:2005 spécifie une méthode enzymatique pour la détermination de la teneur en acide lactique et en lactates, applicable à tous les types de laits secs.

Mleko v prahu - Določevanje vsebnosti mlečne kisline in laktatov (ISO 8069:2005)

General Information

Status
Withdrawn
Publication Date
29-Sep-2007
Withdrawal Date
30-Dec-2007
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
20-Jun-2007
Due Date
17-Oct-2007
Completion Date
20-Jun-2007

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Standards Content (Sample)

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.

---------------------- Page: 1 ----------------------
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.
---------------------- Page: 2 ----------------------
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.
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INTERNATIONAL ISO
STANDARD 8069
IDF
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

address below.
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Published in Switzerland
ii © ISO and IDF 2005 – All rights reserved
---------------------- Page: 5 ----------------------
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.

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
...

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