ISO 17997-1:2004

INTERNATIONAL ISO
STANDARD 17997-1
IDF
29-1
First edition
2004-09-15
Milk — Determination of casein-nitrogen
content —
Part 1:
Indirect method (Reference method)
Lait — Détermination de la teneur en azote de caséine —
Partie 1: Méthode indirecte (Méthode de référence)

Reference numbers
IDF 29-1:2004(E)
©
ISO and IDF 2004
IDF 29-1:2004(E)
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ii © ISO and IDF 2004 – All rights reserved

IDF 29-1:2004(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 17997-1IDF 29-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee
SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC
International. It is being published jointly by ISO and IDF and separately by AOAC International.
ISO 17997IDF 29 consists of the following parts, under the general title Milk — Determination of casein-
nitrogen content:
 Part 1: Indirect method (Reference method)
 Part 2: Direct method
IDF 29-1:2004(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 and AOAC International 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
IDF National Committees casting a vote.
ISO 17797-1IDF 29-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee
SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC
International. It is being published jointly by ISO and IDF and separately by AOAC International.
All work was carried out by the Joint ISO/IDF/AOAC Action Team on Nitrogen compounds, of the Standing
Committee on Main components in milk.
This edition of ISO 17997-1IDF 29-1, together with ISO 17997-2IDF 29-2, cancels and replaces the first
edition of IDF 29:1964, which has been technically revised.
ISO 17997IDF 29 consists of the following parts, under the general title Milk — Determination of casein-
nitrogen content:
 Part 1: Indirect method (Reference method)
 Part 2: Direct method
iv © ISO and IDF 2004 – All rights reserved

IDF 29-1:2004(E)
Introduction
This part of ISO 17997IDF 29 is a classical reference method for the indirect determination of the casein-
nitrogen content of milk. No collaborative study data were available for this method when publishing the first
edition of IDF 29:1964.
Recent research has been completed to develop a better defined indirect reference method. A routine method
for the direct measurement of the casein-nitrogen content of milk is given in ISO 17997-2IDF 29-2. Both parts
of ISO 17997IDF 29 have been collaboratively studied and a reference to the obtained precision data is now
included in each part.
INTERNATIONAL STANDARD
IDF 29-1:2004(E)
Milk — Determination of casein-nitrogen content —
Part 1:
Indirect method (Reference method)
WARNING — The use of the method and equipment described in this standard may involve hazardous
materials, operations and equipment. This standard does not purport to address all the safety risks
associated with its use. It is the responsibility of the user of this standard to establish appropriate
safety and health practices and determine the applicability of local regulatory limitations prior to use.
1 Scope
This part of ISO 17997IDF 29 specifies a reference method for the indirect determination of the casein-
nitrogen content of bovine milk.
The method can be modified for milk from other species or liquid dairy products.
NOTE Casein nitrogen will decrease with milk storage time due to casein breakdown even at 4 °C. The casein
nitrogen of heat-treated milk will be artificially high because of whey-protein denaturation.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 648:1977, Laboratory glassware — One-mark pipettes
ISO 1042:1998, Laboratory glassware — One-mark volumetric flasks
ISO 8968-1IDF 20-1, Milk — Determination of nitrogen content — Part 1: Kjeldahl method
ISO 8968-2IDF 20-2, Milk — Determination of nitrogen content — Part 2: Block-digestion method (Macro
method)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
non-casein-nitrogen content
mass fraction of substances determined according to the procedures specified in this part of
ISO 17997IDF 29
IDF 29-1:2004(E)
3.2
casein-nitrogen content
mass fraction of substances determined according to the procedures specified in this part of
ISO 17997IDF 29
NOTE Both the non-casein-nitrogen content and the casein-nitrogen content are expressed as a mass fraction in
percent.
4 Principle
The total-nitrogen content of a test sample is determined by the method of either ISO 8968-1IDF 20-1 or
ISO 8968-2IDF 20-2. Casein is precipitated from a separate test portion of the same milk by the addition of
acetic acid and sodium acetate solutions, such that the final pH of the mixture is approximately 4,6. The
precipitated milk casein is removed by filtration, so the remaining filtrate contains the non-casein-nitrogen
components. The nitrogen content of the filtrate is determined by the procedure described in
ISO 8968-1IDF 20-1 or ISO 8968-2IDF 20-2. The casein-nitrogen content is calculated as the difference
between the total-nitrogen content and the non-casein-nitrogen content of the milk.
5 Reagents
Use only reagents of recognized analytical grade and glass-distilled water or water of at least equivalent purity.
5.1 Reagents for determination of total nitrogen.
Use the reagents specified in ISO 8968-1IDF 20-1 or ISO 8968-2IDF 20-2.
5.2 Acetic acid solution, c(CH CO H) = 1,75 mol/l.
3 2
Using a volumetric pipette (6.6), add 10,0 ml of glacial acetic acid in a 100 ml volumetric flask (6.3). Dilute to
the mark with water.
5.3 Sodium acetate solution, c(CH CO Na) = 1 mol/l.
3 2
Dissolve 8,20 g of sodium acetate or 13,60 g of sodium acetate trihydrate in water in a 100 ml volumetric flask
(6.3). Dilute to the mark with water.
The sodium acetate solution may be stored at room temperature for one week or at between 0 °C and 4 °C for
6 months.
6 Apparatus
Usual laboratory equipment and, in particular, the following.
6.1 Apparatus for determination of total nitrogen.
Use that specified in ISO 8968-1IDF 20-1 or ISO 8968-2IDF 20-2.
6.2 Water bath, capable of maintaining a temperature of 38 °C to 40 °C.
6.3 One-mark volumetric flasks, with stoppers, of capacity 100 ml, conforming to ISO 1042:1998, class A.
6.4 Bottle-top dispenser, capable of delivering 75 ml of water (optional).
6.5 Graduated measuring cylinder, of capacity up to 100 ml.
2 © ISO and IDF 2004 – All rights reserved

IDF 29-1:2004(E)
6.6 One-mark volumetric pipettes, of capacity 1 ml, 10 ml and 50 ml, conforming to ISO 648:1977,
class A.
6.7 Filter funnel, made of glass or plastic, of diameter 75 mm.
1)
6.8 Filter paper, of diameter 15 cm, nitrogen-free (e.g. Whatman No. 1 or equivalent).
Pleat before use.
6.9 Conical flasks, or equivalent, of capacity 100 ml.
6.10 Analytical balance, capable of weighing to the nearest 0,1 mg.
6.11 Automatic pipettor or (adjustable) micropipette, capable of delivering 1,0 ml (optional).
7 Sampling
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changed during transport or storage.
Sampling is not part of the method specified in this part of ISO 17997IDF 29. A recommended sampling
method is given in ISO 707.
8 Preparation of test sample
Warm the test sample in the water bath (6.2) set at between 38 °C and 40 °C to melt the milk fat so that a
representative test portion of milk can be removed from the test sample. Gently mix the sample immediately
prior to removal and weighing of the test portion (see 9.1 and 9.2).
9 Procedure
9.1 Determination of total nitrogen
Determine the total-nitrogen content of the test sample (Clause 8), w , expressed as a mass fraction in
N
percent, by using the procedure as described in ISO 8968-1IDF 20-1 or ISO 8968-2IDF 20-2.
9.2 Determination of non-casein nitrogen
9.2.1 Test portion
9.2.1.1 Weigh, to the nearest 0,1 mg, approximately 10 g of the prepared test sample (Clause 8) into a
100 ml volumetric flask (6.3). Immediately add 75 ml of water, preheated to 38 °C, using a bottle-top dispenser
(6.4) or a measuring cylinder (6.5).
Additional samples may be weighed and water added at this point. However, care shall be taken to finish step
9.2.1.2 within 30 min after adding the test portion to the volumetric flask.
NOTE This 30-min time limit is to minimize proteolytic degradation of casein during sample preparation.

1) Whatman No. 1 is an example of a product available commercially.
This information is given for the convenience of users of this part of ISO 17997|IDF 29 and does not constitute an
endorsement by ISO or IDF of this product.
IDF 29-1:2004(E)
9.2.1.2 Using a volumetric pipette (6.6) or an automatic pipettor or (adjustable) micropipette (6.11), add
1 ml of acetic acid solution (5.2) to the flask. Stopper the flask and swirl to mix the contents. Place the flask for
10 min in the water bath (6.2) set at between 38 °C and 40 °C.
Using another volumetric pipette (6.6) or an automatic pipettor or (adjustable) micropipette (6.11), add 1 ml of
sodium acetate solution (5.3) to the flask and swirl to mix. Cool the flask and its contents to 20 °C. Dilute to
the mark with water at 20 °C. Stopper the flask and invert to mix.
Let the precipitate settle. Filter the contents of the flask using a filter funnel (6.7) and a pleated filter paper
(6.8). Collect the entire filtrate in a 100 ml conical flask (6.9). The filtrate shall be clear and free of particulate
matter. If not, repeat the procedure starting from 9.2.1 with a new test portion.
NOTE This method uses a fixed volume addition of acetic acid and sodium acetate solutions for every sample. This
will not achieve an exact pH of 4,6 for every milk sample. However, it is a practical compromise that has been used
traditionally for analysis of bovine milks, particularly when a large number of samples are to be analysed. pH variation in
the range between 4,5 and 5,0 has been shown to have a negligible influence on the final result (see Reference [8]). The
alternative is to monitor the pH as acid is added to the sample with the appropriate temperature adjustments for pH-meter
calibration. In this case, the exact dilution of each test portion should be measured and a different dilution factor should be
taken into account for each test portion analysed.�
9.2.1.3 Swirl the clear filt
...