ISO 13875:2005

Liquid milk — Determination of acid-soluble beta-lactoglobulin content — Reverse-phase HPLC method

ISO 13875|IDF 178:2005 specifies a method for the quantitative determination of the beta-lactoglobulin content, soluble at pH 4,6, in liquid milk. The method has been tested over a range between 0 mg and 3 500 mg of beta-lactoglobulin per litre of milk. It is suitable for distinguishing different categories of heat-treated liquid milk.

Lait liquide — Détermination de la teneur en bêta-lactoglobuline soluble dans l'acide — Méthode par chromatographie liquide haute performance en phase inverse

General Information

• Status: Published
• Publication Date: 04-Jan-2005
• ICS: 67.100.10 - Milk and processed milk products
• Technical Committee: ISO/TC 34/SC 5 - Milk and milk products
• Drafting Committee: ISO/TC 34/SC 5 - Milk and milk products
• Current Stage: 9093 - International Standard confirmed
• Completion Date: 13-Jan-2022

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 13875
IDF
178
First edition
2005-02-01


Liquid milk — Determination of acid-
soluble β-lactoglobulin content —
Reverse-phase HPLC method
Lait liquide — Détermination de la teneur en
β-lactoglobuline soluble dans l'acide — Méthode par chromatographie
liquide haute performance en phase inverse




Reference numbers
ISO 13875:2005(E)
IDF 178:2005(E)
©
ISO and IDF 2005

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ISO 13875:2005(E)
IDF 178:2005(E)
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©  ISO and IDF 2005
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ISO 13875:2005(E)
IDF 178:2005(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle . 1
5 Reagents . 1
6 Apparatus. 2
7 Sampling . 3
8 Procedure. 3
8.1 Preparation of test portion. 3
8.2 Preparation of test solution . 4
8.3 Preparation of reference portion . 4
8.4 Preparation of reference solutions for the multi-point calibration . 4
8.5 Preparation of the reference solution for the “single-point” calibration procedure . 5
8.6 HPLC determination. 5
8.7 Integration mode . 6
9 Calculation and expression of results . 8
9.1 Multi-point calibration. 8
9.2 Single-point calibration . 9
9.3 Expression of results. 9
10 Standardization of the reference sample. 10
10.1 General. 10
10.2 Preparation of the standard sample. 10
10.3 Determination of protein content . 10
10.4 Determination of ββββ-LG content in the reference sample. 10
10.5 Calculation of the ββββ-LG content. 10
10.6 Expression of results. 11
11 Precision . 11
11.1 Interlaboratory test . 11
11.2 Repeatability. 11
11.3 Reproducibility . 11
12 Test report. 12
Annex A (informative) Results of interlaboratory trials. 13
Bibliography . 14

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ISO 13875:2005(E)
IDF 178:2005(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 13875IDF 178 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.

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ISO 13875:2005(E)
IDF 178:2005(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 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
the 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 13875IDF 178 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, Characterization of heat treatment, of the
Standing Committee on Minor components and characterization of physical properties, under the aegis of its
project leader, Prof. L. Pellegrino (IT).

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ISO 13875:2005(E)
INTERNATIONAL STANDARD
IDF 178:2005(E)

Liquid milk — Determination of acid-soluble
β-lactoglobulin content — Reverse-phase HPLC method
1 Scope
This International Standard specifies a method for the quantitative determination of the β-lactoglobulin content,
soluble at pH 4,6, in liquid milk. The method has been tested over a range between 0 mg and 3 500 mg of
β-lactoglobulin per litre of milk. It is suitable for distinguishing different categories of heat-treated liquid milk.
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 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
ββββ-lactoglobulin content
ββββ-LG content
mass fraction of substance determined by the procedure specified in this International Standard
NOTE It is expressed in milligrams per litre of test sample.
4 Principle
Casein and denatured whey protein are precipitated isoelectrically from milk at pH 4,6. The acid whey is
separated by centrifuging and filtering. The acid-soluble β-LG content in the acid whey is determined by
reverse-phase HPLC. The soluble β-LG content in the test sample is quantified by single-point or a multi-point
calibration using a reference sample.
5 Reagents
Use only reagents of recognized analytical grade and distilled water or water of at least equivalent purity,
unless otherwise specified.
5.1 Standard sample, pure β-lactoglobulin (A+B genetic variants).
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ISO 13875:2005(E)
IDF 178:2005(E)
Test the chromatographic purity of the β-LG standard sample by the HPLC procedure described in Clause 10.
Determine its precise concentration as described in 10.2.
5.2 Reference sample
The reference sample is reconstituted from a freeze-dried raw bulk milk sample which was originally prepared
from a skimmed raw bulk milk sample. It contains a known amount of soluble β-LG (A+B), which is determined
by the HPLC procedure described in Clause 10.
The freeze-dried reference sample may be stored at 4 °C for 6 months, preventing hydration.
5.3 Reagents for sample preparation
5.3.1 Hydrochloric acid, dilute, c(HCl) = 2 mol/l.
5.3.2 Phosphate buffer solution, of pH 6,7 (final concentration 0,1 mol/l).
Add 57 ml of 0,2 mol/l sodium dihydrogen orthophosphate (NaH PO ) solution to a 200 ml volumetric flask
2 4
(6.10). Add 43 ml of 0,2 mol/l disodium hydrogen orthophosphate (Na HPO ) solution and mix the phosphate
2 4
solutions. Dilute to the mark with water and mix again.
5.4 HPLC elution solvents
Use elution solvents prepared from reagents of recognized HPLC-grade.
SAFETY PRECAUTIONS — Take appropriate safety precautions when handling the elution solvents as
the chemicals may be carcinogenic.
5.4.1 Water, of HPLC-grade.
Laboratory-prepared water may be not sufficiently pure. Impure water produces column contamination and
loss of resolution. If impure or improperly stored trifluoracetic acid is used, peaks can be unresolved or absent
from the chromatogram.
5.4.2 Acetonitrile (CH CN).
3
5.4.3 Trifluoracetic acid (CF COOH), of the highest purity.
3
6 Apparatus
Usual laboratory equipment and, in particular, the following.
6.1 pH-meter, calibrated over the pH range 4,0 to 7,0, and accurate to 0,1 pH units.
6.2 Centrifuge, capable of operating at 2 000 g.
6.3 Centrifuge glass tubes, of capacity about 30 ml.
6.4 Glass vials, of capacity about 5 ml.
6.5 Glass funnels, of diameter about 7 cm.
6.6 Filter paper, fast grade, of diameter about 11 cm.
6.7 Glass test tubes, of capacity about 30 ml.
6.8 One-mark pipettes, capable of delivering 1 ml, 2 ml and 5 ml.
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ISO 13875:2005(E)
IDF 178:2005(E)
6.9 Beakers, of capacities 50 ml and 100 ml.
6.10 One-mark volumetric flasks, of capacities 10 ml, 20 ml, 25 ml, 50 ml and 200 ml.
6.11 Microfiltration tools.
6.11.1 Glass syringe, of capacity 5 ml.
6.11.2 Disposable syringe filter units, of pore size 0,22 µm, used with aqueous solutions.
6.12 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg.
6.13 Magnetic stirrer.
6.14 HPLC equipment.
6.14.1 Elution gradient pumping system, capable of operating at 1,0 ml/min at 200 bar.
6.14.2 Manual or automatic injector, capable of injecting 20 µl.
6.14.3 Column heater, capable of maintaining the column at 40 °C ± 1°C.
6.14.4 UV detector, capable of operating at 205 nm or at 280 nm wavelength and 0,1 AUFS.
6.14.5 Integrator or data-reprocessing software, capable of measuring peak areas.
1)
6.14.6 PLRP-S column , of length 150 mm and internal diameter 4,6 mm, of particle size 5 µm or 8 µm, and
pore size 30 nm; or an equivalent column packed with underivatized polystyrene divinyl benzene, giving an
equivalent chromatographic pattern.
7 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.
8 Procedure
8.1 Preparation of test portion
8.1.1 Check that the reported expiry date of the test sample has not been passed. Bring the closed package
of the test sample to 20 °C ± 2 °C. Just before opening, shake the package and its contents carefully by
inversion.
Open the package and transfer about 50 ml of test sample to a 100 ml beaker (6.9).
The test sample package should not be opened until just before starting the preparation.

1) PLRP-S column is the trade name of a product supplied by Polymer Laboratories Ltd, Church Stretton, United Kingdom.
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. Equivalent products may be used if they can be shown to lead to the same
results.
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ISO 13875:2005(E)
IDF 178:2005(E)
8.1.2 Adjust the pH of the test portion to 4,6 by dropwise addition of the dilute hydrochloric acid (5.3.1) while
stirring continuously. Allow the test portion to stand for 20 min at room temperature.
Transfer the prepared test portion to a centrifuge tube (6.3) and centrifuge at 2 000 g for 20 min. Filter the
supernatant through filter paper (6.6), collecting the casein-free acid whey in a test tube (6.7).
The undiluted acid whey test portion may be stored for 24 h at 4 °C or for 2 weeks at −18 °C. Once defrosted,
the acid whey test portion shall not be refrozen.
8.2 Preparation of test solution
After defrosting, carefully mix the acid whey test portion. Using a pipette (6.8), transfer suitable amounts of the
acid whey test portion, depending on the type of test sample, to a 10 ml volumetric flask (6.10):
a) 1 ml, if from a test sample of raw or pasteurized or high-temperature pasteurized milk (final dilution 1:10);
b) 2 ml, if prepared from a test sample of UHT milk (final dilution 1:5);
c) 5 ml, if prepared from
...