SIST EN ISO 20541:2008

SLOVENSKI STANDARD
SIST EN ISO 20541:2008
01-december-2008
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Milk and milk products - Determination of nitrate content - Method by enzymatic
reduction and molecular-absorption spectrometry after Griess reaction (ISO 20541:2008)
Milch und Milcherzeugnisse - Bestimmung des Nitratgehaltes - Verfahren mit
enzymatischer Reduktion und Molekülabsorptionsspektrometrie nach Griess-Reaktion
(ISO 20541:2008)
Lait et produits laitiers - Détermination de la teneur en nitrates - Méthode par réduction
enzymatique et spectrométrie d'absorption moléculaire après réaction de Griess (ISO
20541:2008)
Ta slovenski standard je istoveten z: EN ISO 20541:2008
ICS:
67.100.01 0OHNRLQPOHþQLSURL]YRGLQD Milk and milk products in
VSORãQR general
SIST EN ISO 20541:2008 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 20541:2008

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SIST EN ISO 20541:2008
EUROPEAN STANDARD
EN ISO 20541
NORME EUROPÉENNE
EUROPÄISCHE NORM
September 2008
ICS 67.100.01

English Version
Milk and milk products - Determination of nitrate content -
Method by enzymatic reduction and molecular-absorption
spectrometry after Griess reaction (ISO 20541:2008)
Lait et produits laitiers - Détermination de la teneur en Milch und Milcherzeugnisse - Bestimmung des
nitrates - Méthode par réduction enzymatique et Nitratgehaltes - Verfahren mit enzymatischer Reduktion
spectrométrie d'absorption moléculaire après réaction de und Molekülabsorptionsspektrometrie nach Griess-
Griess (ISO 20541:2008) Reaktion (ISO 20541:2008)
This European Standard was approved by CEN on 6 August 2008.
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
© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20541:2008: E
worldwide for CEN national Members.

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SIST EN ISO 20541:2008
EN ISO 20541:2008 (E)
Contents Page
Foreword.3

2

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SIST EN ISO 20541:2008
EN ISO 20541:2008 (E)
Foreword
This document (EN ISO 20541:2008) has been prepared by Technical Committee ISO/TC 34 "Agricultural
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 March 2009, and conflicting national standards shall be withdrawn at
the latest by March 2009.
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.
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 the United Kingdom.
Endorsement notice
The text of ISO 20541:2008 has been approved by CEN as a EN ISO 20541:2008 without any modification.

3

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SIST EN ISO 20541:2008

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SIST EN ISO 20541:2008


INTERNATIONAL ISO
STANDARD 20541
IDF
197
First edition
2008-09-01


Milk and milk products — Determination
of nitrate content — Method by enzymatic
reduction and molecular-absorption
spectrometry after Griess reaction
Lait et produits laitiers — Détermination de la teneur en nitrates —
Méthode par réduction enzymatique et spectrométrie d'absorption
moléculaire après réaction de Griess





Reference numbers
ISO 20541:2008(E)
IDF 197:2008(E)
©
ISO and IDF 2008

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SIST EN ISO 20541:2008
ISO 20541:2008(E)
IDF 197:2008(E)
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©  ISO and IDF 2008
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 2008 – All rights reserved

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SIST EN ISO 20541:2008
ISO 20541:2008(E)
IDF 197:2008(E)
Contents Page
Foreword. iv
Foreword. v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle. 2
5 Reagents. 2
6 Apparatus . 4
7 Sampling. 5
8 Preparation of test sample. 5
8.1 Dried milk, dried whey and milk protein concentrates . 5
8.2 Caseins and caseinates . 5
8.3 Cheese . 6
8.4 Whey cheese . 6
9 Procedure . 6
9.1 Preparation of the test portion . 6
9.2 Removal of fat and protein . 6
9.3 Reagent blank test. 7
9.4 Determination. 7
9.5 Calibration . 9
10 Calculation and expression of results. 9
10.1 Nitrite (matrix blank) content (see Clause 4, Note 3). 9
10.2 Total nitrite/nitrate content . 9
10.3 Nitrate content. 10
11 Precision. 10
11.1 Interlaboratory testing. 10
11.2 Repeatability. 10
11.3 Reproducibility. 11
12 Test report . 11
Annex A (informative) Interlaboratory trials . 12
Bibliography . 15

© ISO and IDF 2008 – All rights reserved iii

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SIST EN ISO 20541:2008
ISO 20541:2008(E)
IDF 197:2008(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 20541⎪IDF 197 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.

iv © ISO and IDF 2008 – All rights reserved

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SIST EN ISO 20541:2008
ISO 20541:2008(E)
IDF 197:2008(E)
Foreword
IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide.
IDF membership comprises National Committees in every member country as well as regional dairy
associations having signed a formal agreement on cooperation with IDF. All members of IDF have 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 20541⎪IDF 197 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 Minor compounds of the Standing Committee on
Minor components and characterization of physical properties under the aegis of its project leaders,
Mr. M. Carl (DE) and Mrs. C. Bäckman (FL).

© ISO and IDF 2008 – All rights reserved v

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SIST EN ISO 20541:2008

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SIST EN ISO 20541:2008
ISO 20541:2008(E)
INTERNATIONAL STANDARD
IDF 197:2008(E)

Milk and milk products — Determination of nitrate content —
Method by enzymatic reduction and molecular-absorption
spectrometry after Griess reaction
WARNING — The use of this International Standard may involve hazardous materials, operations and
equipment. This standard does not purport to address all the safety problems associated with its use.
It is the responsibility of the user of this standard to establish safety and health practices and
determine the applicability of regulatory limitations prior to use.
1 Scope
This International Standard specifies a method for the determination of the nitrate content of milk and milk
products by molecular-absorption spectrometry after Griess reaction (preceded by enzymatic reduction).
The method is, in particular, applicable to whole, partly skimmed, skimmed and dried milk, hard, semi-hard
and soft cheeses, processed cheese, whey cheese, caseins, caseinates, dried whey and milk protein
concentrates.
The method can be used at contents corresponding to a measured concentration in the sample solution (with
blank subtracted) of more than 0,2 mg/l.
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 last edition of the referenced document
(including any amendments) applies.
ISO 565, Test sieves — Metal wire cloth, perforated metal plate and electroformed sheet — Nominal sizes of
openings
ISO 648, Laboratory glassware — Single-volume pipettes
ISO 835, Laboratory glassware — Graduated pipettes
ISO 1042, Laboratory glassware — One-mark volumetric flasks
ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
nitrite content
mass fraction of nitrite compounds determined by the procedure specified in this International Standard
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SIST EN ISO 20541:2008
ISO 20541:2008(E)
IDF 197:2008(E)
3.2
nitrate content
mass fraction of nitrate compounds determined by the procedure specified in this International Standard
−
NOTE The nitrate content is expressed as the mass fraction in milligrams of nitrate ion (NO ) per kilogram of
3
product.
4 Principle
A test portion is dispersed in warm water. The fat and proteins are removed either by precipitation using
Carrez reagents and filtering or by centrifugal ultra-filtration using membrane cones (see Notes 1 and 2). The
nitrate is reduced to nitrite in a portion of the filtrate by means of nitrate reductase. A red-violet azo dye is
developed, in portions of both the unreduced filtrate (for nitrite) and the reduced solution (for nitrate), by
addition of sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride, and the absorbance measured
at a wavelength of 540 nm (or Hg 546 nm). The nitrite content of the sample and the total nitrite content after
reduction of nitrate are calculated by comparing the measured absorbances with those of a set of sodium
nitrite calibration solutions. The nitrate content is calculated from the difference between these two contents.
NOTE 1 The two alternative procedures for removal of fat and protein are described in 9.2.1 and 9.2.2.
NOTE 2 For whey powder, whey protein concentrate and similar products, ultra-filtration is used in preference to
Carrez precipitation as the latter often leads to turbidity problems and, as a consequence, to poor precision.
NOTE 3 The low endogenous nitrite level is not reported but taken into account in the matrix blank solution.
5 Reagents
Unless otherwise specified, use only reagents of recognized analytical grade, free of nitrate and nitrite, and
water complying with ISO 3696 grade 3 at least, free from nitrate and nitrite. Water used for preparation of
enzyme or coenzyme solutions shall be freshly double-distilled or of equivalent purity.
5.1 Sodium hydroxide solution, c(NaOH) = 1 mol/l.
5.2 Sodium chloride solution, c(NaCI) = 0,9 g/100 ml.
5.3 Hydrochloric acid, ρ (HCI) = 1,19 g/ml.
20
5.4 Hydrochloric acid solution, c(HCI) = 2 mol/l.
Carefully add 160 ml of hydrochloric acid (5.3) to about 700 ml of water in a 1 000 ml one-mark volumetric
flask (6.4) while regularly swirling the contents. Cool the contents to room temperature. Dilute to the mark with
water and mix carefully.
5.5 Carrez reagents, as follows:
5.5.1 Carrez reagent I: Potassium hexacyanoferrate(II) solution, c(K [Fe(CN) ].3H O) = 150 g/l.
4 6 2
Dissolve 15,0 g of potassium hexacyanoferrate(II) trihydrate in water in a 100 ml one-mark volumetric flask
(6.4). Dilute to the mark with water and mix.
5.5.2 Carrez reagent II: Zinc sulfate solution, c(ZnSO .7H O) = 300 g/l.
4 2
Dissolve 30,0 g of zinc sulfate heptahydrate in water in a 100 ml one-mark volumetric flask (6.4). Dilute to the
mark with water and mix.
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SIST EN ISO 20541:2008
ISO 20541:2008(E)
IDF 197:2008(E)
5.6 Standard solutions, as follows:
5.6.1 Sodium nitrite (NaNO ) stock solution.
2
Accurately weigh (75,0 ± 0,1) mg of pre-dried (at 102 °C for 2 h) sodium nitrite into a 100 ml one-mark
volumetric flask. Dissolve it in a suitable amount of water. Dilute to the mark with water and mix. The nitrite
stock solution obtained contains 500 mg of nitrite per litre.
Prepare calibration solutions by diluting the stock solution with water to give several solutions with different
nitrite concentrations in the range from 0,05 mg/I to 5,0 mg/I.
When stored at room temperature, the sodium nitrite stock solution remains stable for 1 day.
5.6.2 Potassium nitrate (KNO ) stock solution.
3
Accurately weigh (81,5 ± 0,1) mg of pre-dried (at 102 °C for 2 h) potassium nitrate into a 100 ml one-mark
volumetric flask. Dissolve it in a suitable amount of water. Dilute to the 100 ml mark with water and mix. The
obtained nitrate stock solution contains 500 mg of nitrate per litre.
Prepare calibration solutions by diluting the stock solution with water to give several solutions with different
nitrate concentrations in the range from 0,05 mg/I to 5,0 mg/I.
When stored at 4 °C, the potassium nitrate stock solution remains stable for 1 week.
5.7 Potassium phosphate buffer solution, pH = 7,5.
Accurately weigh (57,6 ± 0,1) mg of dipotassium hydrogen phosphate (K HPO .3H O) into a 100 ml one-mark
2 4 2
volumetric flask. Dissolve it in a suitable amount of water. Dilute to the mark with water and mix.
Accurately weigh (17,0 ± 0,1) mg of potassium dihydrogen phosphate (KH PO ) into a 50 ml one-mark
2 4
volumetric flask. Dissolve it in a suitable amount of water. Dilute to the mark with water and mix.
Using the pH-measurement unit (6.18), adjust the pH of the dipotassium hydrogen phospate solution to pH 7,5
by addition of potassium dihydrogen phosphate solution.
When stored at 4 °C, the potassium phosphate buffer solution remains stable for 2 weeks.
5.8 NADPH/FAD solution.
Weigh accurately (5,6 ± 0,1) mg of 3-nicotinamide-adenine dinucleotide phosphate (reduced form),
tetrasodium salt (β-NADPH-Na , with a mass fraction of at least 98 %), and (80,0 ± 0,1) mg flavine-adenine
4
dinucleotide, disodium salt (FAD-Na , with a mass fraction of at least 88 %), into a 25 ml one-mark volumetric
2
flask.
Dissolve them in a suitable amount of potassium phosphate buffer solution (5.7). Dilute to the mark with the
buffer solution (5.7) and mix.
Freshly prepare the NADPH/FAD solution immediately before use.
5.9 Nitrate reductase (NR) solution.
Weigh 65 mg of nitrate reductase (NR) from Aspergillus niger (EC 1.6.6.2, Iyophilizate containing
approximately 0,4 U/mg) into a 10 ml measuring tube. Add 5 ml of water and mix.
When stored at 4 °C, the nitrate reductase solution remains stable for 2 weeks.
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SIST EN ISO 20541:2008
ISO 20541:2008(E)
IDF 197:2008(E)
5.10 Colour reagents, as follows:
5.10.1 Colour reagent solution I: Sulfanilamide (NH C H SO NH ).
2 6 4 2 2
Weigh 400 mg of sulfanilamide into a 50 ml one-mark volumetric flask (6.4). Dissolve it, heating on a water-
bath if necessary, in hydrochloric acid solution (5.4).
Cool the solution to room temperature. Dilute to the mark with the hydrochloric acid solution (5.4) and mix. If
necessary, filter the reagent solution thus obtained.
When stored at 4 °C, colour reagent solution I remains stable for 4 weeks.
5.10.2 Colour reagent solution II: N-(1-Naphthyl)ethylenediamine dihydrochloride
(C H NHCH CH NH .2HCI).
10 7 2 2 2
Weigh 50 mg of N-(1-naphthyl)ethylenediamine dihydrochloride into a 50 ml one-mark volumetric flask (6.4).
Dissolve it in a suitable amount of water.
Dilute to the 50 ml mark with water and mix. If necessary, filter the solution thus obtained.
When stored at 4 °C, colour reagent solution II remains stable for 4 weeks.
5.11 Reagent kits are also commercially available. Carefully follow the instructions of this International
Standard when using such kits (in particular in the case of 5.8).
6 Apparatus
Clean all glassware thoroughly and rinse with water to ensure that it is free from nitrate and nitrite.
Usual laboratory equipment and, in particular, the following:
6.1 Analytical balance, capable of weighing to the nearest 0,1 mg.
6.2 Sample container, with an airtight Iid.
6.3 Conical flasks, of capacities 100 ml, 500 ml and 1 000 ml, with ground-glass stoppers.
6.4 One-mark volumetric flasks, of nominal capacities 25 ml, 50 ml, 100 ml and 1 000 ml, complying with
the requirements of ISO 1042, class A.
6.5 Pipettes, capable of delivering 1 ml, 2 ml, 5 ml and 10 ml, respectively, complying with the
requirements of ISO 648, class A, or ISO 835. Where appropriate, burettes may be used instead of pipettes.
6.6 Graduated pipettes, of the partial-delivery type, as used in enzyme tests.
6.7 Graduated cylinders, of capacities 20 ml and 50 ml.
6.8 Beakers, of capacities 20 ml and 50 ml.
6.9 Centrifuge, with cooling device, capable of centrifuging cups (6.10) and membrane cones (6.21) with a
centrifugal acceleration of 3 000g.
6.10 Centrifuge cups, of diameter 15 mm.
6.11 Membrane filter, of pore size 0,45 µm, for use with a syringe.
6.12 Glass funnel, of suitable diameter.
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SIST EN ISO 20541:2008
ISO 20541:2008(E)
IDF 197:2008(E)
6.13 Spectrometer, suitable for measuring absorbance at a wavelength of 540 nm, or spectral line
photometer with a mercury lamp and filter, suitable for measuring absorbance at a wavelength of 546 nm.
6.14 Optical cells, semi-micro type (disposable or glass cuvettes), of optical path length 1 cm.
6.15 Grinding device, suitable for grinding the test sample, if necessary. To avoid loss of moisture, the
device shall not produce undue heat.
6.16 Test sieve, of woven wire cloth, of diameter 200 mm, with openings of nominal size 500 µm and a
receiver complying with the requirements of ISO 565.
6.17 Magnetic stirrer.
6.18 pH-measurement unit, consisting of a pH-meter and glass/reference electrodes, capable of measuring
at 20 °C.
6.19 Water bath, with shaking facility, capable of operating at (70 ± 0,5) °C.
6.20 Hotplate.
6.21 Membrane cones, MWCO 5 000 D, capacity 4 ml, for centrifugal ultra-filtration of the sample solution.
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 receives a sample that is truly representative and has not been damaged or
changed during transport or storage.
Store the laboratory sample in such a way that deterioration and change in composition are prevented.
8 Preparation of test sample
8.1 Dried milk, dried whey and milk protein concentrates
Transfer the test sample to a sample container (6.2) of capacity about twice the volume of the test sample.
Close the container immediately. Mix the test sample thoroughly by repeatedly shaking and inverting the
container.
8.2 Caseins and caseinates
8.2.1 Thoroughly mix the test sample, if necessary after transferring all of it to a sample container (6.2) of
suitable capacity, by repeatedly shaking and inverting the container.
8.2.2 Transfer 50 g of the test sample to a test sieve (6.16). If the 50 g portion completely, or nearly
completely, passes through the sieve, pass the whole mixed test sample (see 8.2.1) through the sieve. If the
test sample does not pass completely through the sieve, use the grinding device (6.15) to ensure that it does.
Immediately transfer the entire sieved test sample to a sample container (6.2). Mix thoroughly in the closed
container. During these operations, take precautions to avoid any change in the water content of the product.
After the test sample has been prepared, proceed with the preparation of the test portion (see 9.1) as soon as
possible.
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SIST EN ISO 20541:2008
ISO 20541:2008(E)
IDF 197:2008(E)
8.3 Cheese
8.3.1 Prior to the analysis, remove any rind or mouldy surface layer from the test sample so as to provide a
test sample representative of the cheese as it is usually consumed.
8.3.2 Grind the test sample by means of a suitable device (6.15). Mix the ground mass quickly and, if
possible, grind a second time and again mix thoroughly. Clean the grinding device after grinding each sample.
If the test sample cannot be ground, mix it thoroughly by intensive stirring and kneading.
8.3.3 As soon as possible after grinding, transfer the test sample to a sample container (6.2) to await the
determination, which should preferably be carried out without delay. If delay is unav
...