SIST EN ISO 8968-1:2014

SLOVENSKI STANDARD
SIST EN ISO 8968-1:2014
01-april-2014
1DGRPHãþD
SIST EN ISO 8968-1:2002
SIST EN ISO 8968-2:2002
0OHNRLQPOHþQLSURL]YRGL'RORþHYDQMHYVHEQRVWLGXãLNDGHO0HWRGDSR
.MHOGDKOXLQL]UDþXQVXURYLKEHOMDNRYLQ,62
Milk and milk products - Determination of nitrogen content - Part 1: Kjeldahl principle and
crude protein calculation (ISO 8968-1:2014)
Milch und Milcherzeugnisse - Bestimmung des Stickstoffgehaltes - Teil 1: Kjeldahl-
Verfahren und Berechnung des Rohproteingehaltes (ISO 8968-1:2014)
Lait et produites laitieres - Détermination de la teneur en azote et calcul de protéine brute
- Partie 1: Méthode Kjeldahl (ISO/FDIS 8968-1:2013)
Ta slovenski standard je istoveten z: EN ISO 8968-1:2014
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
SIST EN ISO 8968-1:2014 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 8968-1:2014

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SIST EN ISO 8968-1:2014

EUROPEAN STANDARD
EN ISO 8968-1

NORME EUROPÉENNE

EUROPÄISCHE NORM
February 2014
ICS 67.100.10 Supersedes EN ISO 8968-1:2001, EN ISO 8968-2:2001
English Version
Milk and milk products - Determination of nitrogen content - Part
1: Kjeldahl principle and crude protein calculation (ISO 8968-
1:2014)
Lait et produits laitiers - Détermination de la teneur en azote Milch und Milcherzeugnisse - Bestimmung des
- Partie 1: Méthode Kjeldahl et calcul de la teneur en Stickstoffgehaltes - Teil 1: Kjeldahl-Verfahren und
protéines brutes (ISO 8968-1:2014) Berechnung des Rohproteingehaltes (ISO 8968-1:2014)
This European Standard was approved by CEN on 18 January 2014.

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-CENELEC 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-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 8968-1:2014 E
worldwide for CEN national Members.

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SIST EN ISO 8968-1:2014
EN ISO 8968-1:2014 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 8968-1:2014
EN ISO 8968-1:2014 (E)
Foreword
This document (EN ISO 8968-1:2014) has been prepared by Technical Committee ISO/TC 34 “Food
products” in collaboration with 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 August 2014, and conflicting national standards shall be withdrawn at
the latest by August 2014.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 8968-1:2001, EN ISO 8968-2:2001.
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, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 8968-1:2014 has been approved by CEN as EN ISO 8968-1:2014 without any modification.


3

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SIST EN ISO 8968-1:2014

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SIST EN ISO 8968-1:2014
INTERNATIONAL ISO
STANDARD 8968-1
IDF
20-1
Second edition
2014-02-01
Milk and milk products —
Determination of nitrogen content —
Part 1:
Kjeldahl principle and crude protein
calculation
Lait et produits laitiers — Détermination de la teneur en azote —
Partie 1: Méthode Kjeldahl et calcul de la teneur en protéines brutes
Reference numbers
ISO 8968-1:2014(E)
IDF 20-1:2014(E)
©
ISO and IDF 2014

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SIST EN ISO 8968-1:2014
ISO 8968-1:2014(E)
IDF 20-1:2014(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO/IDF 2014
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or
by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written
permission. Permission can be requested from either ISO or IDF at the respective address below or ISO’s member body in the
country of the requester.
ISO copyright office International Dairy Federation
Case postale 56 • CH-1211 Geneva 20 Silver Building • Bd Auguste Reyers 70/B • B-1030 Brussels
Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88
Fax + 41 22 749 09 47 Fax + 32 2 733 04 13
E-mail copyright@iso.org E-mail info@fil-idf.org
Web www.iso.org Web www.fil-idf.org
Published in Switzerland
ii © ISO and IDF 2014 – All rights reserved

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SIST EN ISO 8968-1:2014
ISO 8968-1:2014(E)
IDF 20-1:2014(E)
Contents Page
Forewords .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 2
6 Apparatus . 3
7 Sampling . 5
8 Preparation of test sample . 5
8.1 Whole, partially skimmed or skimmed liquid milk . 5
8.2 Hard, semi-hard and processed cheese . 5
8.3 Dried milk and dried milk products . 5
9 Procedures . 6
9.1 Traditional method . 6
9.2 Block digestion method . 7
9.3 Blank test . 9
9.4 Recovery tests .10
10 Calculation and expression of results .11
10.1 Calculation .11
10.2 Expression of results .12
11 Precision .13
11.1 Interlaboratory tests .13
11.2 Liquid milk, whole milk and skimmed milk .13
11.3 Hard, semi-hard and processed cheese .14
11.4 Dried milk and dried milk products .14
12 Test report .14
Annex A
(informative)

Test portion .15
Annex B
(informative)

Collaborative trials .16
Bibliography .18
© ISO and IDF 2014 – All rights reserved iii

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SIST EN ISO 8968-1:2014
ISO 8968-1:2014(E)
IDF 20-1:2014(E)
Forewords
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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2. www.iso.org/directives
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received. www.iso.org/patents
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 34, Food and food products, Subcommittee SC 5,
Milk and milk products and the International Dairy Federation (IDF) and is being published jointly by ISO
and IDF.
This second edition of ISO 8968-1|IDF 20-1 cancels and replaces the first edition of ISO 8968-1|
IDF 20-1:2001, ISO 8968-2|IDF 20-2:2001, ISO 5549:1978/IDF 92:1979 and ISO/TS 17837| IDF/RM 25:2008
which have been technically revised.
iv © ISO and IDF 2014 – All rights reserved

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SIST EN ISO 8968-1:2014
ISO 8968-1:2014(E)
IDF 20-1:2014(E)
The International Dairy Federation (IDF) 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 Standing Committees are circulated to the National
Committees for voting. Publication as an International Standard requires approval by at least 50 % of
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.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
ISO 8968-1|IDF 20-1 was prepared by the International Dairy Federation and Technical Committee
ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by ISO
and IDF.
The work was carried out by the IDF-ISO Project Group on Nitrogen, of the Standing Committee on
Analytical Methods for Composition (SCAMC), under the aegis of its project leaders: Mr. R. Johnson (NZ),
Mr. J. Romero (US), Dr. Barbano (US), Dr. Orlandini (IT), and Mr. Psathas (CY).
This second edition of ISO 8968-1|IDF 20-1 cancels and replaces the first edition of ISO 8968-1|
IDF 20-1:2001, ISO 8968-2|IDF 20-2:2001, ISO 5549:1978/IDF 92:1979 and ISO/TS 17837| IDF/RM 25:2008
which have been technically revised.
© ISO and IDF 2014 – All rights reserved v

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SIST EN ISO 8968-1:2014

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SIST EN ISO 8968-1:2014
ISO 8968-1:2014(E)
INTERNATIONAL STANDARD
IDF 20-1:2014(E)
Milk and milk products — Determination of nitrogen
content —
Part 1:
Kjeldahl principle and crude protein calculation
WARNING — The use of this International Standard might involve the use of hazardous materials,
operations, and equipment. This International Standard does not purport to address all the safety
risks associated with its use. It is the responsibility of the user of this International Standard
to establish appropriate safety and health practices and determine the applicability of local
regulatory limitations prior to use.
1 Scope
This International Standard specifies a method for the determination of the nitrogen content and crude
protein calculation of milk and milk products by the Kjeldahl principle, using traditional and block
digestion methods.
The methods are applicable to:
— liquid cow’s (whole, partially skimmed or skimmed milk), goat’s and sheep’s whole milk;
— hard, semi-hard and processed cheese;
— dried milk and dried milk products (including milk-based infant formulae, milk protein concentrate,
whey protein concentrate, casein and caseinate).
The methods are not applicable to samples containing ammonium caseinate.
NOTE Inaccurate crude protein results will be obtained if non-milk sources of nitrogen are present in the
products specified in this International Standard.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable to its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 385, Laboratory glassware — Burettes
ISO 8655-3, Piston-operated volumetric apparatus — Part 3: Piston burettes
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
nitrogen content
mass fraction of nitrogen determined by the specified procedure
Note 1 to entry: It is expressed as a percentage.
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SIST EN ISO 8968-1:2014
ISO 8968-1:2014(E)
IDF 20-1:2014(E)
3.2
crude protein content
mass fraction of crude protein calculated as specified
Note 1 to entry: It is expressed as a percentage.
4 Principle
A test portion is digested with a mixture of concentrated sulfuric acid and potassium sulfate. Using
copper sulfate (II) as a catalyst to thereby convert any organic nitrogen present to ammonium sulfate.
The function of the potassium sulfate is to elevate the boiling point of the sulfuric acid and to provide
a stronger oxidizing mixture for digestion. Excess sodium hydroxide is added to the cooled digest to
liberate ammonia. The liberated ammonia is steam distilled into the excess boric acid solution and
titration with hydrochloric acid standard volumetric solution is carried out. The nitrogen content is
calculated from the amount of ammonia produced.
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or
demineralized water or water of equivalent purity.
NOTE The solutions specified in this procedure might be different than those required for the operation of
automated titrators. An effort is made to address those, but it is the responsibility of the operator to follow the
directions of the equipment manufacturer.
5.1 Potassium sulfate (K SO ), nitrogen free.
2 4
5.2 Copper (II) sulfate pentahydrate solution, c(CuSO .5H O) = 5,0 g/100 ml.
4 2
Dissolve 5,0 g of copper(II) sulfate pentahydrate in water in a 100 ml one-mark volumetric flask. Dilute
to the mark with water and mix.
5.3 Sulfuric acid (H SO ), with a mass fraction of between 95 % and 98 %, nitrogen-free (approximately
2 4
ρ = 1,84 g/ml).
20
5.4 Sodium hydroxide (NaOH) solution, nitrogen-free, containing 50 g of sodium hydroxide per
100 g.
With automated distillation systems, other mass fractions of sodium hydroxide may be used, provided
an excess of sodium hydroxide is dispensed to the distillation mixture; for example, a mass fraction of
40 % sodium hydroxide solution may be used instead of a mass fraction of 50 %, where plugging of the
automated flow system is a problem. The total volume of such sodium hydroxide solution should be
considered in order to maintain the suitable distillation volumes.
5.5 Indicator solution
Dissolve 0,1 g of methyl red in 95 % (volume fraction) ethanol in a 50 ml one-mark volumetric flask
(6.16). Dilute to 50 ml with ethanol and mix. Dissolve 0,5 g of bromocresol green in 95 % (volume
fraction) ethanol in a 250 ml one-mark volumetric flask (6.16). Dilute to 250 ml with ethanol and mix.
Mix one part of the methyl red solution with five parts of the bromocresol green solution or combine and
mix all of both solutions.
5.6 Boric acid solution, c(H BO ) = 40,0 g/l.
3 3
Dissolve 40,0 g of boric acid (H BO ) in 1 l of hot water in a 1 000 ml one-mark volumetric flask (6.16).
3 3
Allow the flask and its contents to cool to 20 °C. Adjust to the mark with water, add 3 ml of the indicator
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SIST EN ISO 8968-1:2014
ISO 8968-1:2014(E)
IDF 20-1:2014(E)
solution (5.5) and mix. Store the solution, which will be light orange in colour, in a borosilicate glass
bottle. Protect the solution from light and sources of ammonia fume during storage.
With automated distillation systems, other boric acid concentrations may be used after validating
accordingly.
If using the electronic pH end point titration, the addition of the indicator solution to the boric acid
solution may be omitted. On the other hand, the change in colour may also be used as a check on proper
titration procedures.
5.7 Hydrochloric acid standard volumetric solution, c(HCI) = (0,1 ± 0,0005) mol/l.
It is recommended to purchase this material prestandardized by the manufacturer, which meets, or
exceeds, these specifications. Often, the systematic errors (which can be avoided) introduced by an
analyst diluting a concentrated stock acid and then determining the molarity of the acid, cause poor
reproducibility performance of the method in this part. The analyst should not use a solution for titration
that has a higher concentration than 0,1 mol/l, because this will reduce the total titration volume per
sample and the uncertainty in readability of the burette will become a larger percentage of the value.
This will have a negative impact on the method repeatability and reproducibility performance.
If sulfuric acid is substituted for hydrochloric acid, the solution should have a concentration of
0,05 ± 0,000 3 mol/l.
5.8 Ammonium sulfate [(NH ) SO ], minimum assay 99,9 % (mass fraction) on dried material.
4 2 4
Immediately before use, dry the ammonium sulfate at 102 °C ± 2 °C for not less than 2 h. Cool to room
temperature in a desiccator.
5.9 Tryptophan (C H N O ) or lysine hydrochloride (C H ClN O ), minimum assay 99 % (mass
11 12 2 2 6 15 2 2
fraction).
Do not dry these reagents in an oven before use.
5.10 Sucrose, with a mass fraction of nitrogen of not more than 0,002 %.
Do not dry the sucrose in an oven before use.
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 Water bath, capable of maintaining a water temperature between 38 °C and 40 °C.
6.2 Analytical balance, capable of weighing to the nearest 0,1 mg.
6.3 Burette or automatic pipette, capable for delivering 1,0 ml portions of the copper sulfate solution
(5.2).
6.4 Graduated measuring cylinders, of capacity 25 ml, 50 ml, 100 ml and 500 ml.
6.5 Conical flasks, of capacity 500 ml.
6.6 Automatic burette, of suitable capacity e.g. 20 ml, with resolution of at least 0,004 ml, complying
with the requirements of ISO 8655-3. Alternatively, a burette, of capacity 50 ml, graduated at least at
every 0,1 ml, complying with the requirements of ISO 385, class A may be used for the analysis of milk.
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SIST EN ISO 8968-1:2014
ISO 8968-1:2014(E)
IDF 20-1:2014(E)
NOTE The manual burette does not have sufficient resolution to achieve the required number of significant
figures for all other products.
6.7 Grinding device
6.8 Digestion flasks (Kjeldahl), of 500 ml or 800 ml capacities. Suitable to the digestion system to be
used and to the specifications of the manufacturer of the digestion apparatus (6.10 or 6.11).
6.9 Boiling aids, e.g. hard pieces of porcelain or high-purity amphoteric alundum (i.e. carbarundum)
granules, plain, mesh size 10. Do not reuse the boiling aids.
NOTE Glass beads of approximately 5 mm diameter can also be used, but they might not promote as efficient
boiling as the alundum granules and more foaming problems can be encountered during digestion with glass
beads.
6.10 Digestion apparatus, to hold the digestion flasks (6.8) in an inclined position (approximately
45 °), with electric heaters or gas burners, which do not heat the flasks above the level of their contents,
and with a fume extraction system.
The heater source should be adjustable to control the maximum heater setting to be used during
digestion. Preheat the heat source at the heater setting for evaluation. In the case of a gas heater, the
preheating period shall be 10 min and for an electric heater, it shall be 30 min. For each of the heaters,
determine the heater setting that brings 250 ml of water, including 5 to 10 boiling aids with an initial
temperature of 25 °C, to its boiling point in 5 min to 6 min. This is the maximum heater setting to be
used during digestion.
6.11 Distillation apparatus (traditional method), made of borosilicate glass or other suitable material
to which can be fitted a digestion flask (6.8) consisting of an efficient splash-head connected to an efficient
condenser with straight inner tube and an outlet tube attached to its lower end. The connecting tubing
and stopper(s) shall be close-fitting and preferably made of polychloroprene.
1)
NOTE The distillation apparatus mentioned above can be replaced by the complete Parnas-Wagner
distillation configuration or other suitable equipment.
6.12 Digestion block (block digesting method), aluminium alloy block or equivalent block, fitted with
an adjustable temperature control and device for measuring block temperature.
6.13 Digestion tubes (block digesting method), of 250 ml in capacity, suitable for use with the
digestion block (6.12).
6.14 Exhaust manifold (block digesting method), suitable for use with the digestion tubes (6.13).
6.15 Centrifugal scrubber apparatus or filter pump or aspirator (block digesting method),
constructed of acid-resistant material, for use with mains water supply.
6.16 Volumetric flasks, one mark of 50 ml, 250 ml and 1 000 ml capacities.
6.17 Distillation unit (block digesting method), capable of steam distilling, manual or semi–automatic,
suited to accept the 250 ml digestion tubes (6.13) and the 500 ml conical flasks (6.5).
1) Parnas-Wagner is an example of glassware configuration utilized for Kjeldahl distillation available commercially.
This information is given for the convenience of users and does not constitute an endorsement by either ISO or IDF
of this product.
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SIST EN ISO 8968-1:2014
ISO 8968-1:2014(E)
IDF 20-1:2014(E)
6.18 Automatic titrator provided with a pH-meter.
The pH-meter should be calibrated properly in the range of pH 4 to pH 7 following normal laboratory
pH-calibration procedures. The automatic titrator burette shall comply with the requirements of 6.6.
6.19 Spatula or suitable transfer device.
6.20 Filter paper, nitrogen–free, of dimensions and porosity suitable to hold the cheese test portion.
6.21 Illuminated magnetic stirrer plate.
7 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling
method is given in ISO 707|IDF 50.
It is important that the laboratory receive a sample which is representative and has not been damaged
or changed during transport or storage.
8 Preparation of test sample
8.1 Whole, partially skimmed or skimmed liquid milk
Place the test sample in the water bath (6.1) set at 38 °C to 40 °C. Mix gently by inversion without causing
frothing or churning. Once the sample is mixed thoroughly, cool to room temperature.
Proceed as indicated in 9.1 or 9.2.
8.2 Hard, semi-hard and processed cheese
Remove the rind, smear or mouldy surface layer of the cheese, in such a way as to provide a
...