ISO 23318:2022

INTERNATIONAL ISO
STANDARD 23318
IDF 249
First edition
2022-11
Milk, dried milk products and
cream — Determination of fat content
— Gravimetric method
Lait, produits laitiers secs et crème — Détermination de la teneur en
matière grasse — Méthode gravimétrique
Reference numbers
IDF 249:2022(E)
IDF 249:2022(E)
© ISO and IDF 2022
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Published in Switzerland
ii
IDF 249:2022(E)
Contents Page
Foreword .v
1 S c op e . 1
2 Nor m at i ve r ef er enc e s . 2
3 Terms and definitions . 2
4 P r i nc iple . 2
5 R e a g ent s . 2
6 A pp a r at u s . 3
7 S a mpl i n g . 4
8 P reparation of test sample . 5
8 .1 M i lk . 5
8.2 D ried milk products . 5
8 . 3 E v ap or at e d m i l k . 5
8.4 Sweetened condensed milk . 5
8.5 W hey cheese . 6
8.6 Cream . 6
8.7 S kimmed milk, whey and buttermilk . 6
8.8 M ilk-based infant foods . 6
8.8.1 Liquid products . 6
8.8.2 Viscous or pasty products . 7
8.9 M ilk-based edible ices and ice mixes . 7
8.9.1 E dible ices, ice mixes and concentrated ice mixes . 7
8.9.2 D ried ice mixes . 7
9 P r o c e du re .7
9.1 Te s t p or t ion . 7
9.2 B lank tests . 8
9.3 P reparation of fat-collecting vessel. 8
9.4 D e t er m i n at ion . 9
9.4.1 Preparation steps . 9
9.4 . 2 D e t er m i n at ion . 10
10 C alculation and expression of results .13
10 .1 C a lc u l at ion . 13
10.2 E xpression of results . .13
11 P r e c i s ion .14
11.1 I nterlaboratory test . . 14
11.2 R epeatability . 14
11.3 Reproducibility . 14
12 Te s t r ep or t .15
Annex A (informative) Additional procedures .16
Annex B (informative) Fat-extraction tube model with siphon or wash-bottle fittings .18
Annex C (informative) Interlaboratory study on raw milk .19
Annex D (informative) Interlaboratory study on raw sheep milk and raw goat milk .21
Annex E (informative) Interlaboratory study on dried milk products .22
Annex F (informative) Interlaboratory study on cream .23
Annex G (informative) Interlaboratory study on skimmed milk (specific method when high
accuracy is required, see 9.1) .24
Annex H (informative) Interlaboratory study on other dairy products .25
iii
IDF 249:2022(E)
Bibliography .27
iv
IDF 249:2022(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.
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 (see 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 (see 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 of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document 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.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
IDF 249:2022(E)
IDF (the International Dairy Federation) is a non-profit private sector organization representing the
interests of various stakeholders in dairying at the global level. IDF members are organized in National
Committees, which are national associations composed of representatives of dairy-related national
interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and
governments/food control authorities.
ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis
and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International
Standards using the logos and reference numbers of both organizations.
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. 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 (see 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.
This document was prepared by the IDF Standing Committee on Analytical Methods for Composition and
ISO 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 ISO/IDF Action Team on C32 of the Standing Committee on Analytical
Methods for Composition under the aegis of its project leader, Mr P. Trossat (FR).
vi
INTERNATIONAL STANDARD
IDF 249:2022(E)
Milk, dried milk products and cream — Determination of
fat content — Gravimetric method
WARNING — The use of this document may involve hazardous materials, operations and
equipment. This document does not purport to address all the safety problems associated
with its use. It is the responsibility of the user of this document to establish safety and health
practices and determine the applicability of regulatory limitations prior to use.
1 S cope
This document specifies the method for the determination of fat content.
The method is applicable to:
a) raw milk (cow, sheep, goat), reduced fat milk, skimmed milk, chemically preserved milk and
processed liquid milk;
b) dried milk products (e.g. whole, partially skimmed, skimmed milk powder; dairy permeate powder;
whey powder; blend skimmed milk powder and vegetable fat; milk based infant formula powder);
c) raw, processed and sour cream.
For the following products, the precision figures are given in Annex H. These precision figures are
derived from interlaboratory studies not conforming to the requirements from ISO 5725-2 in terms of
number of samples (< 6) and number of participating laboratories (< 8).
d) evaporated milk and sweetened condensed milk (e.g. liquid sweetened and unsweetened
concentrated milk);
e) whey cheese as defined in CODEX CXS 284-1999;
f) liquid whey and buttermilk;
g) milk-based edible ices and ice mixes;
h) liquid concentrated infant foods.
The method does not apply in the following cases:
— For b), when the powder contains hard lumps which do not dissolve in ammonia solution. This is
noticeable by a distinct smell and the result of the determination will be too low. In such cases, a
method using the Weibull-Berntrop principle is suitable, e.g. ISO 8262-3|IDF 124-3.
— For c), The method is not applicable to sour creams with starch or other thickening agents. When
separation or breakdown of fat occurs, a method using the Weibull-Berntrop principle is suitable,
e.g. ISO 8262-3|IDF 124-3.
— For e), to products which do not dissolve completely in ammonia solution, as the result of the
determination will be too low. With such products, a method using the Weibull-Berntrop principle
is suitable, e.g. ISO 8262-3|IDF 124-3.
— For g), to milk-based edible ices and ice mixes in which the level of emulsifier, stabilizer or thickening
agent or of egg yolk or of fruits, or of combinations of these constituents, makes the Röse-Gottlieb
method unsuitable. With such products, a method using the Weibull-Berntrop principle is suitable,
e.g. ISO 8262-2|IDF 124-2.
IDF 249:2022(E)
— For h), to products which do not dissolve completely in ammonia due to the presence of starch or
dextrin at mass fractions of more than 5 % (in dry matter), or to the presence of hard lumps. For
such products, a method using the Weibull-Berntrop principle is suitable, e.g. ISO 8262-1|IDF 124-1.
2 Normat ive references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 835, Laboratory glassware — Graduated pipettes
ISO 1042, Laboratory glassware — One-mark volumetric flasks
ISO 3889|IDF 219, Milk and milk products — Specification of Mojonnier-type fat extraction flasks
ISO 4788, Laboratory glassware — Graduated measuring cylinders
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
fat content
mass fraction of substances
Note 1 to entry: Fat content is determined by the procedure specified in this document.
Note 2 to entry: The fat content is expressed as a percentage by mass.
4 Principle
An ammoniacal ethanolic solution of a test portion is extracted with diethyl ether and light petroleum.
The solvents are removed by distillation or evaporation. The mass of the substances extracted is
determined.
NOTE This principle is usually described as the Röse-Gottlieb principle.
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and only distilled or
demineralized water or water of equivalent purity.
The reagents shall leave no appreciable residue when the determination is carried out by the method
specified (see Annex A).
5.1 Ammonia solution, containing a mass fraction of NH of approximately 25 %.
If ammonia solution of this concentration is not available, a more concentrated solution of known
concentration may be used (see 9.4.2.1).
IDF 249:2022(E)
5.2 Ethanol (C H OH), or ethanol denatured by methanol, containing a volume fraction of ethanol of
2 5
at least 94 %, see Clause A.5.
5.3 Indicator solution (optional). The use of indicator solutions allows the interface between the
solvent and aqueous layers to be seen more clearly (see 9.4.2.3). Other aqueous indicator solutions can
be used provided that they do not affect the result of the determination.
5.3.1 Bromocresol purple solution, mass concentration 1 g/100 ml. Dissolve 1 g of bromocresol
purple in 10 ml of ethanol and dilute to 100 ml with water.
5.3.2 Patent blue V. Dissolve 1 g of patent blue V (sodium salt) in water and dilute to 100 ml with
water.
5.4 Diethyl ether (C H OC H ), free from peroxides (see Clause A.3), containing no more than 7 mg/
2 5 2 5
kg of antioxidants, and conforming to the requirements for the blank test (see Clauses A.1 and A.4).
WARNING — The use of diethyl ether can lead to hazardous situations. Observe current safety
precautions for handling, use and disposal.
5.5 Light petroleum, with any boiling range between 30 °C and 60 °C or, as equivalent, pentane
(CH (CH ) CH ) with a boiling point of 36 °C and conforming to the requirements for the blank test (see
3 2 3 3
Clauses A.1 and A.4).
The use of pentane is recommended because of its higher purity and consistent quality.
NOTE Petroleum ether and petroleum benzine with an appropriate boiling range are some suitable
commercial names of this reagent.
5.6 Mixed solvent.
Shortly before use, mix equal volumes of diethyl ether (5.4) and light petroleum (5.5).
6 Apparatus
The usual laboratory equipment and, in particular, the following shall be used.
Use graduated pipettes in accordance with ISO 835, one-mark volumetric flasks in accordance with
ISO 1042 and graduated measuring cylinders in accordance with ISO 4788.
6.1 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg.
6.2 Centrifuge, capable of holding the fat-extraction flasks or tubes (6.6) and capable to produce a
radial acceleration of around 80g to 90g at the outer end of the flasks or tubes.
The use of the centrifuge is optional but recommended (see 9.4.2.6).
6.3 Distillation or evaporation apparatus, for distilling the solvents and ethanol from the boiling
or conical flasks, or evaporating from beakers and dishes (see 9.4.2.12) at a temperature not exceeding
100 °C.
6.4 Drying oven, electrically heated, with ventilation port(s) fully open, capable of being maintained
at a temperature of 102 °C ± 2 °C throughout its working space. The oven shall be fitted with a suitable
thermometer.
6.5 Water bath, capable of being maintained at a temperature of 37,5 °C ± 2,5 °C, 50 °C ± 5 °C,
65 °C ± 5 °C and at boiling point.
IDF 249:2022(E)
6.6 Fat-extraction flasks:
6.6.1 Mojonnier-type fat-extraction flasks, as specified in ISO 3889|IDF 219.
6.6.2 Extraction tubes-type fat extraction flask. It is also possible to use fat extraction tubes with
siphon or wash-bottle fittings (see the model given in Annex B).
6.6.3 Stoppers. The flasks or tubes shall be provided with stoppers of different material [bark cork,
silicone rubber, polytetrafluoroethylene (PTFE) or glass] unaffected by the reagents used. Bark corks
shall be washed with diethyl ether (5.4), kept in water at 60 °C or more for at least 15 min, and shall
then be allowed to cool in the water so that they are saturated when used.
6.7 Rack, for holding the fat-extraction flasks (or tubes) (6.6).
6.8 Wash bottle, suitable for use with the mixed solvent (5.6). A plastic wash bottle shall not be used.
6.9 Fat-collecting vessels, e.g.:
— boiling flasks, flat-bottomed, of capacity 125 ml to 250 ml;
— conical flasks, of capacity 250 ml;
— metal dishes.
If metal dishes are used, they shall be of stainless steel, flat-bottomed with a diameter of 80 mm to
100 mm and a height of approximately 50 mm.
6.10 Measuring cylinders or delivering systems, compatible with the use of solvents of capacities
5 ml and 25 ml.
6.11 Pipettes or delivering systems, of capacity 10 ml.
6.12 Tongs, made of metal, for holding flasks, beakers or dishes.
6.13 Blender, fitted with a bowl with a capacity of 1 l with its lid or any other device suitable for
preparing the test sample.
6.14 Boiling aids (optional), fat free in non-porous porcelain, silicon carbide or glass.
7 S ampling
A representative sample should be sent to the laboratory. It should not be damaged or changed during
transport or storage.
Sampling is not part of the method specified in this document. A recommended sampling method is
given in ISO 707|IDF 50.
From the time of sampling to the time of commencing the procedure, store laboratory liquid samples at
a temperature of 4 °C ± 2 °C and dried products at room temperature. For evaporated milk, sweetened
condensed milk and milk-based infant foods, store samples in sealed cans or bottles unopened at a
temperature below 20 °C, until the time of starting the procedure.
IDF 249:2022(E)
8 Pr eparation of test sample
8.1 Milk
Using the water bath (6.5), warm the test sample to a temperature of 38 °C ± 2 °C. Gently mix the
test sample thoroughly without causing frothing or churning. Then cool the test sample quickly to
approximately 20 °C.
If a homogeneous test sample can be obtained without pre-warming (e.g. for samples of skimmed milk),
bring the test sample to a temperature of 20 °C ± 2 °C and gently mix thoroughly by repeatedly inverting
the sample bottle.
A reliable value for the fat content cannot be expected:
a) if the milk is churned;
b) when a distinct smell of free fatty acids is perceptible;
NOTE Goat milk naturally contains a low level of free fatty acids, which are not completely extracted in
this method.
c) if, during or after preparation of the test sample, white particles are visible on the walls of the
sample bottle or fat droplets float on the surface of the sample.
8.2 Dried milk pr oducts
Thoroughly mix the test sample by repeatedly rotating and inverting the sample container. If necessary,
transfer all the test sample to an airtight container of approximately twice the volume of the test sample
to allow this operation to be carried out.
8.3 Evaporated milk
Shake and invert the sample container. Open the sample container and pour the sample slowly into
a second sample container, provided with an airtight lid. Mix by repeated transfer, taking care to
incorporate in the sample any fat or other constituent adhering to the wall and ends of the first
container. Finally, transfer the product as completely as possible to the second container.
If necessary in the case of samples in sealed cans, condition the unopened container in the water bath
(6.5) maintained at a temperature of 50 °C ± 5 °C. Remove and shake the can vigorously every 15 min.
After 2 h, remove the can and allow it to cool to room temperature.
Remove the lid entirely and thoroughly mix the sample by stirring with a spoon or spatula. If fat
separates, do not test the sample.
8.4 S weetened condensed milk
Open the sample container and mix thoroughly with a spoon or spatula. Use an up-and-down rotary
movement in such a way that the top layers and the content of the lower corners of the container are
moved and mixed. Take care to incorporate in the sample any milk adhering to the wall and ends of the
container. Transfer the sample as completely as possible to a second sample container, provided with an
airtight lid. Close the second container.
If necessary, in the case of samples in sealed cans, condition the unopened can in the water bath
(6.5) at a temperature of 38 °C ± 2 °C. Open the can, scrape out all milk adhering to the interior of
the can, transfer to a dish large enough to permit stirring thoroughly and mix until the whole mass is
homogeneous.
In the case of a sample in a collapsible tube, open the tube and transfer the contents to a jar. Then cut
the tube and scrape out all material adhering to the interior and add to the contents of the jar.
IDF 249:2022(E)
8.5 Whey cheese
Prepare the test sample using an appropriate device. Quickly mix the ground or grated mass and, if
possible, grind it a second time. Again mix thoroughly. Clean the device after preparing each test sample.
If the test sample cannot be ground or grated, mix it thoroughly by intensive kneading, e.g. with a pestle
in a mortar. The risk of moisture loss during grinding or grating of the sample should be avoided as far
as practically possible.
Keep the prepared test sample in an airtight sample container until the time of analysis, which should
be carried out on the same day. If delay is unavoidable, take every precaution to ensure proper storage
of the test sample. When refrigerated, ensure that any condensation of moisture on the inside surface of
the container is thoroughly and uniformly reincorporated into the test sample.
8.6 Cream
Warm the test sample to a temperature of 38 °C ± 2 °C by means of the water bath (6.5), if necessary.
Thoroughly, but gently, mix the test sample by repeatedly inverting the sample bottle, or, if the cream
is very thick, by stirring with a spatula, without causing frothing or churning, and cool quickly to
approximately 20 °C.
Churned cream should not be cooled as it has to be weighed at a temperature of between 30 °C and
40 °C (see 9.1).
NOTE A reliable value for the fat content cannot be expected when a distinct smell of free fatty acids is
perceptible, and/or if during or after preparation of the test sample white particles are visible on the walls of
the sample bottle or fat droplets float on the surface of the sample. In such cases, a method utilizing the Weibull-
Berntrop principle is suitable, such as ISO 8262-3|IDF 124-3.
8.7 Skimmed milk, w hey and buttermilk
Warm the test sample to a temperature of 38 °C ± 2 °C by means of the water bath (6.5), if necessary.
Gently mix the test sample thoroughly by repeatedly inverting the sample bottle without causing
frothing or churning. Cool the test sample quickly to approximately 20 °C.
NOTE A reliable value for the fat content cannot be expected if the milk is churned, when a distinct smell of
free fatty acids is perceptible and/or if during or after preparation of the test sample white particles are visible
on the walls of the sample bottle or fat droplets float on the surface of the sample.
8.8 Milk -based infant foods
8.8.1 Liquid products
Shake and invert the sample container. Open the container to pour the product slowly into a second
sample container provided with an airtight lid. Mix by repeated transfer of the product, taking care
to incorporate in the sample any fat or other constituent adhering to the wall and ends of the first
container. Transfer the test sample as completely as possible to the second sample container. Close this
container.
If necessary, condition the unopened sample container in the water bath (6.5) at a temperature of
50 °C ± 5 °C. Remove and shake the container vigorously every 15 min. After 2 h, remove the container,
dry the outside with a tissue and allow to cool to room temperature. Remove the lid or cap entirely
and thoroughly mix the contents by stirring with a spoon or spatula. If fat separates, do not test the
sample. Transfer the test sample as completely as possible to a second sample container provided with
an airtight lid. Close this container.
IDF 249:2022(E)
8.8.2 Viscous or pasty products
Open the sample container and thoroughly mix the contents with a spoon or spatula. If possible, use an
up-and-down rotary movement in such a way that the top layers and the contents of the lower corners
of the container are moved and mixed. Take care to incorporate in the test sample any fat or other
constituents adhering to the wall and ends of the container. Transfer the test sample as completely as
possible to a second sample container provided with an airtight lid. Close this container.
If necessary, condition the unopened sample container in the water bath (6.5) maintained 38 °C ± 2 °C.
Remove the container, dry the outside with a tissue and open it. Scrape out all test sample adhering to
the interior of the container. Transfer the test sample to a dish large enough to permit thorough stirring
and mix until the whole mass is homogeneous. Transfer the test sample as completely as possible to a
second sample container provided with an airtight lid. Close this container.
8.9 Milk -based edible ices and ice mixes
8.9.1 Edible ices, ice mixes and concentrated ice mixes
8.9.1.1 Do not allow the temperature to exceed 12 °C at any time during the preparation of the (ice)
pieces. Remove any coating of non-ice character from the test sample.
8.9.1.2 If possible, separate the layers of multilayer products, in which the layers possibly have
different fat contents, while the product is still frozen. Prepare individual test samples from each layer
as specified in 8.9.1.3.
8.9.1.3 Cut the test sample into pieces. Select several pieces at random to give a total mass of
approximately 100 g, if possible. Place the pieces in a blender jar. Cover the jar with its lid and allow
the pieces to soften at room temperature. Mix plain test samples for 2 min, and test samples containing
particulate matter (e.g. nuts, hard candy chips) for not more than 7 min, to obtain a homogeneous
mixture.
If fat separates or churning occurs, discard the mixture and repeat the preparation process using a
shorter mixing time. Immediately transfer the mixed test sample to a suitable airtight container and
proceed with the determination within 1 h.
8.9.2 Dried ice mixes
Mix thoroughly by rotating and inverting the sample container. If necessary, transfer the test sample to
a suitable airtight container of adequate capacity to allow mixing. If the test sample still contains lumps
or pieces of ingredients, homogenize it in an appropriate blender (6.13).
9 Procedure
9.1 Test portion
Mix the test sample by gently stirring or rotating and inverting the container several times.
For skimmed milk, whey and buttermilk, two test portions are extracted in two fat-extraction flasks
(6.6). The extracts of the two flasks are poured into one prepared fat-collecting vessel (see 9.3).
Immediately weigh, to the nearest 1 mg, directly or by difference, in a fat-extraction flask (6.6), one of
the test portions as shown in Table 1.
Transfer the test portion in the fat extraction flask as completely as possible into the lower (small) bulb
for the Mojonnier.
IDF 249:2022(E)
Table 1 — Mass of the test portion for the different matrices
Matrix Test portion
Milk, skimmed milk, whey and buttermilk 10 g to 11 g
a) about 1,0 g of dried high-fat milk, of
dried whole milk or of dried butter
serum;
b) about 1,5 g of dried partially skimmed
milk;
Dried milk products
c) about 1,5 g of dried skimmed milk;
d) about 1,5 g of dried whey;
e) about 1,5 g of dried buttermilk.
Evaporated milk 4 g to 5 g
Sweetened condensed milk 2,0 g to 2,5 g
Whey cheese 3,0 g ± 0,2 g
Cream 0,3 g to 0,6 g of extracted fat
1,5 g to 10,0 g corresponding to 1,0 g to 1,5 g
Milk-based infant foods
of dry matter
Edible ices, ice mixes 4 g to 5g
Concentrated ice mixes 2,0 g to 2,5 g
Dried ice mixes 0,9 g to 1,1 g
9.2 Blank tests
Carry out a blank test simultaneously with the determination using the same procedure and the same
reagents, but replacing the test portion in 9.1 by 10 ml of water (see Clause A.2).
When a batch of test samples is analysed, the number of drying cycles may differ between different
samples. If one blank sample is used for the entire batch, ensure that the blank value, used in the
calculation of the fat content of any individual sample, was obtained under the same conditions as the
individual test sample.
If the value obtained in the blank test regularly exceeds 1,0 mg, check the reagents if this has not been
done recently (see Clause A.1). Corrections for values of more than 2,5 mg in the blank test shall be
reported in the test report (see Clause A.2).
9.3 Pr eparation of fat-collecting vessel
Dry a fat-collecting vessel (6.9) with a few boiling aids (6.14) in the oven (6.4) maintained at 102 ± 2 °C
for at least 1 h.
Protect the fat-collecting vessel from dust and allow it to cool to the temperature of the weighing room
(a glass fat-collecting vessel for at least 1 h, a metal dish for at least 30 min).
To avoid insufficient cooling or unduly long cooling times, the fat-collecting vessel should not be placed
in a desiccator.
Use tongs (6.12) to place the fat-collecting vessel on the balance. Weigh the fat-collecting vessel to the
nearest 1,0 mg.
NOTE In particular, the use of tongs effectively avoids temperature variations.
IDF 249:2022(E)
9.4 Determination
9.4.1 Preparation steps
9.4.1.1 Milk, skimmed milk, w hey and buttermilk
Carry out the determination without delay.
9.4.1.2 Dried milk pr oducts
Carry out the determination without delay. Add about 10 ml of preheated water at a temperature of
65 °C ± 5 °C to the test portion in the fat-extraction flask (see Table 1) to obtain a total volume of 10 ml
to 11 ml. Use the water to wash the test portion on to the bottom of the flask (into the small bulb for
Mojonnier type flask). Mix thoroughly with the test portion (in the small bulb for Mojonnier type flask)
until the test portion is completely dispersed.
9.4.1.3 Whey cheese
Carry out the determination without delay. Add about 10 ml of preheated water at a temperature of
65 °C ± 5 °C to the test portion in the fat-extraction flask (see Table 1) to obtain a total volume of 10 ml
to 11 ml. Use the water to wash the test portion on to the bottom of the flask (into the small bulb for
Mojonnier type flask). Mix thoroughly with the test portion (in the small bulb for Mojonnier type flask)
until the test portion is completely dispersed.
Heat the contents of the fat-extraction flask in the boiling water bath (6.5). Shake gently occasionally
until the test portion is completely dispersed. Leave the flask for 20 min in the boiling water bath. Then
cool the flask in running water to room temperature.
9.4.1.4 Evaporated milk
Carry out the determination without delay. Add water at a temperature of about 50 °C to the test portion
in the fat-extraction flask (see Table 1) to obtain a total volume of 10 ml to 11 ml. Use the water to wash
the test portion on to the bottom of the flask (into the small bulb for Mojonnier type flask). Shake gently
with slight warming at about 50 °C in the water bath (6.5) until the test portion is completely dispersed.
Cool in running water to room temperature.
9.4.1.5 Cream
Carry out the determination without delay. Add an amount of water at about 50 °C to the test portion
in the fat-extraction flask (see Table 1) to obtain a total volume of 10 ml to 11 ml. Use the water to
wash the test portion on to the bottom of the flask (into the small bulb for Mojonnier type flask). Mix
thoroughly with the test portion (in the small bulb for Mojonnier type flask). Cool in running water to
room temperature.
9.4.1.6 Milk -based edible ices and ice mixes
Carry out the determination without delay. Add 6 ml, 8 ml or 10 ml of preheated water at a temperature
of 65 °C ± 5 °C to the test portion in the fat-extraction flask (see Table 1) as appropriate to obtain a
total volume of 10 ml to 11 ml. Use the water to wash the test portion on to the bottom of the flask
(into the small bulb for Mojonnier type flask). Mix thoroughly with the test portion (in the small bulb
for Mojonnier type flask). Cool, except for the test portion of dried ice mixes, in running water to room
temperature.
9.4.1.7 Milk -based infant foods
Carry out the determination without delay. If necessary, add preheated water at a temperature of
65 °C ± 5 °C to the test portion in the fat-extraction flask (see Table 1) to obtain a total volume of 10 ml
IDF 249:2022(E)
to 11 ml. Use the water to wash the test portion on to the bottom of the flask (into the small bulb for
Mojonnier type flask). Shake gently with slight warming in a water bath (6.5) maintained at 50 °C ± 5 °C
until the test portion is completely dispersed.
9.4.2 Determination
9.4.2.1 Add 2 ml of ammonia solution (5.1) to the test portion (9.4.1), or an equivalent amount of a
more concentrated ammonia solution (see 5.1). Mix thoroughly with the test portion in the flask (in the
small bulb for Mojonnier type flask).
9.4.2.2 Only when testing dried milk, milk-based infant foods and milk-based edible ices and ice
mixes, close the flask and heat to 65 °C ± 5 °C in the water bath (6.5) for 15 min to 20 min with occasional
shaking. Cool in running water to room temperature.
9.4.2.3 Add 10 ml of ethanol (5.2). Mix gently but thoroughly by allowing the contents of the flask to
flow backwards and forwards and for Mojonnier type flask, between the two bulbs while not bringing
the liquid too near to the neck. If desired, add two drops of the indicator solution (5.3). For whey cheese,
if necessary, cool the flask in running water to room temperature.
9.4.2.4 Add 25 ml of diethyl ether (5.4). Close the fat-extraction flask with a stopper (6.6.3).
Shake the flask vigorously for 1 min, but not so vigorously as to cause formation of a persistent emulsion.
For Mojonnier type flask only, while shaking, keep the flask in a horizontal position with the small bulb
extending upwards, periodically allowing the liquid in the large bulb to run into the small bulb.
If necessary, cool the flask in running water to about room temperature. Carefully remove the bung or
stopper and rinse it and the neck of the flask with a little mixed solvent (5.6). Use the wash bottle (6.8)
so that the rinsings run into the flask.
9.4.2.5 Add 25 ml of light petroleum (5.5). Close the fat-extraction flask with the stopper. Mix gently
again for 30 s as specified in 9.4.2.4.
9.4.2.6 Centrifuge the closed fat-extraction flask for between 1 min and 5 min at a radial acceleration
of around 80g to 90g. If a centrifuge (6.2) is not available, allow the closed flask to stand in the rack
(6.7) for at least 30 min until the supernatant layer is clear and distinctly separated from the aqueous
layer. If necessary, cool the flask in running water, to room temperature.
9.4.2.7 Carefully remove stopper and rinse it and the inside of the neck of the fat-extraction flask
with a little mixed solvent (5.6). Use the wash bottle (6.8) so that the rinsings run into the flask. For
Mojonnier only, if the interface is below the bottom of the stem of the flask, raise it slightly above this
level by gently adding water down the side of the flask (see Figure 1) to facilitate the decanting of
solvent.
For Mojonnier type fat extraction flasks, hold the fat-extraction flask by the small bulb and carefully
decant as much as possible of the supernatant layer into the prepared fat-collecting vessel (see 9.3)
containing a few boiling aids (6.14) (optional with metal dishes). Avoid decanting any of the aqueous
layer (see Figure 2).
NOTE In Figures 1 and 2, one of the three types of fat-extraction flasks (6.6.1) specified in ISO 3889|IDF 219
has been chosen, but this does not imply any preference over other types.
IDF 249:2022(E)
Key
1 solvent
2 interface
3 aqueous layer
a
At second and third extraction.
b
At first extraction.
Figure 1 — Before decanting
Key
1 solvent
2 aqueous layer
a
At second and third extraction.
b
At first extraction.
Figure 2 — After decanting
For extraction tubes type fat extraction flask, insert a siphon or a wash-bottle fitting into the tube (see
Annex B). Push down the long inner limb of the fitting until the inlet is approximately 4 mm above the
interface between the layers. The inner limb of the fitting sh
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