INTERNATIONAL ISO

STANDARD 7305

Third edition
2019-03

Milled cereal products —

Determination of fat acidity
Produits de mouture des céréales — Détermination de l'acidité grasse

Reference number
ISO 7305:2019(E)

© ISO 2019
ISO 7305:2019(E)


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Contents Page

Foreword......................................................................................................................................................................................................................................... iv
Introduction...................................................................................................................................................................................................................................v
1 Scope.................................................................................................................................................................................................................................. 1
2 Normative references....................................................................................................................................................................................... 1
3 Terms and definitions...................................................................................................................................................................................... 1
4 Principle......................................................................................................................................................................................................................... 1
5 Reagents......................................................................................................................................................................................................................... 1
6 Apparatus...................................................................................................................................................................................................................... 2
7 Sampling......................................................................................................................................................................................................................... 2
8 Preparation of test sample.......................................................................................................................................................................... 2
8.1 General............................................................................................................................................................................................................ 2
8.2 Products not requiring grinding............................................................................................................................................... 2
8.3 Products requiring grinding......................................................................................................................................................... 3
9 Determination of moisture content of the test sample................................................................................................. 3
10 Procedure..................................................................................................................................................................................................................... 3
10.1 Number of determinations............................................................................................................................................................ 3
10.2 Test portion................................................................................................................................................................................................. 3
10.3 Determination.......................................................................................................................................................................................... 3
10.4 Blank test...................................................................................................................................................................................................... 3
11 Expression of results......................................................................................................................................................................................... 3
12 Precision........................................................................................................................................................................................................................ 4
12.1 General............................................................................................................................................................................................................ 4
12.2 Repeatability, r ........................................................................................................................................................................................ 4
12.3 Reproducibility, R ................................................................................................................................................................................. 4
12.4 Comparison of two sets of measurements in one laboratory.......................................................................... 5
12.5 Comparison of two sets of measurements in two laboratories..................................................................... 5
12.6 Uncertainty.................................................................................................................................................................................................. 5
12.7 Test report.................................................................................................................................................................................................... 5
Annex A (informative) Results of interlaboratory tests.................................................................................................................... 6
Bibliography................................................................................................................................................................................................................................. 8

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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
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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).
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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 4,
Cereals and pulses.
This third edition cancels and replaces the second edition (ISO 7305:1998), which has been technically
revised. The main changes compared with the previous edition are as follows:
— the indicator has been modified and the procedure relating to laboratory practices has been revised;
— the expression of results has been changed to milligrams of sodium hydroxide per 100 g of dry
matter for more precision.
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.

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Introduction
This document describes a method of estimating the quantity of long-chain, non-esterified fatty acids
which are liberated by the action of lipase during the storage of milled cereal products. It therefore
provides a sensitive and significant test to characterize the state of conservation and the utilization
values of these products.
The solvent used for the extraction, 95 % to 96 % ethanol, breaks all the low-energy links where fatty
acids are involved, and solubilizes the latter rapidly and quantitatively, with the exclusion of the major
part of amino acids and mineral salts.
Observation of the colour change at the end point of the titration is facilitated by the absence of turbidity
in the solution.

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INTERNATIONAL STANDARD ISO 7305:2019(E)

Milled cereal products — Determination of fat acidity

1 Scope
This document specifies a method for the determination of the fat acidity of milled cereal products. It is
applicable to flours and semolinas obtained from wheat and durum wheat, and to pasta.
NOTE This document appears to be applicable also to grains, flours and semolinas obtained from maize, and
rye flour and oat flakes, but a further interlaboratory test is necessary before confirming this extension of the
field of application.

2 Normative 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 712, Cereals and cereal products — Determination of moisture content — Reference method
ISO 12099, Animal feeding stuffs, cereals and milled cereal products — Guidelines for the application of
near infrared spectrometry

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:​//www​.iso​.org/obp
— IEC Electropedia: available at http:​//www​.electropedia​.org/
3.1
fat acidity
quantity of acids, essentially non-esterified fatty acids, extracted according to the procedure described
in this document
Note 1 to entry: Fat acidity is expressed in milligrams of sodium hydroxide per 100 g of dry matter.

Note 2 to entry: A conversion of the result obtained by the calculation in Clause 11 can be done to express the
result in grams of sulphuric acid.

4 Principle
Dissolution of the acids in ethanol at room temperature, centrifuging and titration of an aliquot portion
of the supernatant liquid against sodium hydroxide.

5 Reagents
Use only reagents of recognized analytical grade and distilled or demineralized water or water of
equivalent purity.

5.1 Ethanol, 95 % to 96 % (volume fraction).

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5.2 Sodium hydroxide standard volumetric solution, c (NaOH) = 0,05 mol/l in ethanol (5.1)
(volume fraction), free of carbonates.

The exact concentration shall be known and checked immediately prior to each series of determinations
of fat acidity.
Use a solution prepared at least five days in advance and stored in a brown glass bottle, fitted with a
rubber stopper. The solution shall be colourless or straw coloured.

5.3 Thymolphthalein, indicator solution, 1 g per 100 ml in ethanol (5.1) (volume fraction), changing
from colourless to blue.

6 Apparatus
Usual laboratory apparatus and, in particular, the following.

6.1 Sieves, of wire gauze, of nominal aperture size 160 µm and 500 µm.

6.2 Centrifuge tubes, of capacity 45 to 50 ml, hermetically stoppered.

6.3 Centrifuge, capable of a centrifugal acceleration of 2 000g.

6.4 Pipettes or automatic distributors, of capacities 20 ml and 30 ml.

6.5 Conical flask, of capacity 250 ml.

6.6 Microburette, graduated in 0,01 ml divisions.

6.7 Rotary stirrer, capable of 30 r/min to 60 r/min.

6.8 Analytical balance, capable of weighing to an accuracy of ±0,01 g.

6.9 Grinder, capable of grinding semolina and pasta without any appreciable heating.

7 Sampling
Sampling is not part of the method specified in this document.

8 Preparation of test sample

8.1 General
Acidity increases during storage, therefore the samples shall be stored in sealed bottles at about 4 °C.
Allow the sample to return to laboratory temperature in the sealed bottle before taking test portions.

8.2 Products not requiring grinding

Products in which the particles completely pass a sieve of aperture size 500 µm (6.1) and for which
at least 80 % passes a sieve of aperture size 160 µm (6.1) do not need to be ground before the
determination. Mix the sample well before taking the test portion (10.1).

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8.3 Products requiring grinding

Products not meeting the particle size characteristics mentioned above require grinding.
Grind about 50 g of sample using the grinder (6.9) until the particle size characteristics specified in 8.2
are achieved. Mix well before taking the test portion (10.1).

9 Determination of moisture content of the test sample

Determine the moisture content of the test sample in accordance with the method specified in ISO 712
or using an apparatus utilizing near infrared spectroscopy for which the performances have been
demonstrated in accordance with ISO 12099.

10 Procedure

10.1 Number of determinations

Carry out two single determinations using the samples prepared in accordance with Clause 8.
If the absolute difference between two results is greater than that the repeatability limit defined in
12.2, repeat the determination until the result meets this requirement.

10.2 Test portion

Weigh, to the nearest 0,01 g, approximately 5 g of the test sample (see Clause 8) and place it in a
centrifuge tube (6.2).

10.3 Determination
Using a pipette (6.4), add 30 ml of the ethanol (5.1) to the centrifuge tube (6.2). Seal the tube hermetically
and place it on the rotary stirrer (6.7). Keep stirring for 1 h at room temperature. Then centrifuge (6.3)
for 5 min.
By means of a pipette (6.4), transfer 20 ml of the supernatant liquid to an Erlenmeyer flask (6.5). Add
five drops of Thymolphthalein (5.3).
Titrate the solution, using the microburette (6.6), with the sodium hydroxide solution (5.2) until a blue
colour appears.

10.4 Blank test

Carry out a blank test in parallel with the determination, beginning at 10.3 and replacing the 20 ml of
supernatant liquid by 20 ml of ethanol (5.1).

11 Expression of results
The fat acidity, ANa, expressed in milligrams of sodium hydroxide per 100 g of dry matter, is obtained by
Formula (1):

6 000 (V1 − V0 ) c 100

ANa = × (1)
m 100 − w

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where

c is the exact concentration, expressed in moles per litre, of the standard volumetric sodium
hydroxide solution used;

m is the mass, in grams, of the test portion (10.2);

V1 is the volume, in millilitres, of the sodium hydroxide solution used in the determination (10.3);

V0 is the volume, in millilitres, of the sodium hydroxide solution used in the blank test (10.4);

w is the moisture content, expressed as a percentage by mass, of the test sample (Clause 9);

6 000 is a constant to be applied for sodium hydroxide, i.e. (40 × 1,5 × 100).
Calculate the arithmetic mean of two results meeting the repeatability conditions (see 12.2).
Express the result to the nearest milligram.
To convert the results expressed in milligrams of sodium hydroxide to grams of sulphuric acid, divide
the result obtained by 816.

12 Precision

12.1 General
Details of interlaboratory tests on the precision of the method are summarized in Annex A. The values
derived from these interlaboratory tests may not be applicable to concentration ranges and matrices
other than those given. Table A.2 gives the r values for different levels of fatty acidity.

12.2 Repeatability, r
The absolute difference between two independent single test results, obtained using the same method
on identical test material in the same laboratory by the same operator using the same equipment within
a short interval of time, will in not more than 5 % of cases be greater than the repeatability limit.
r = sr × 2,77

r = (0,009 7x + 0,658 7) × 2,77

where x is the mean fat acidity.

12.3 Reproducibility, R
The absolute difference between two single test results, obtained by using the same method on the same
identical test material in different laboratories with different operators using different equipment.
In practice, it is not appropriate for comparing the results from two laboratories if the test concerned
imposes repeatability conditions.
The appropriate comparison tool is the critical difference as described in 12.4 and 12.5
NOTE The standard deviation of reproducibility (sR = 0,053 2x + 2,520 8, where x is the mean fat acidity)
derived from the interlaboratory tests is a criterion to appreciate the reliability of the uncertainty.

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12.4 Comparison of two sets of measurements in one laboratory

The critical difference between two averaged values, each obtained from two test results, under
repeatability conditions, CDr, is equal to Formula (2):

= 1 , 98 S r = ( 0 , 0097 x + 0 , 658 7 ) × 1 , 98
1 1 1
CDr = 2 , 8 S r + = 2, 8 S r (2)
2n 1 2n 2 2
where

sr is the repeatability standard deviation;

n1 and n2 are the number of test results corresponding to each of the averaged values;

x is the mean fat acidity.

12.5 Comparison of two sets of measurements in two laboratories

The critical difference between two averaged values, each obtained in two different laboratories, from
two test results, under repeatability conditions, CDR , is equal to Formula (3):

1 1
CDR = 2 , 8 s R 2 − s r 2 (1 − − ) = 2, 8 s R 2 − 0 , 5s r 2 (3)
2n 1 2n 2
where

sr is the repeatability standard deviation;

sR is the reproducibility standard deviation;

n1 and n2 are the number of test results corresponding to each of the averaged values.

12.6 Uncertainty
It is possible to evaluate the measurement uncertainties using data obtained from studies conducted
in accordance with ISO 5725-2:1994: the reproducibility standard deviation obtained in a collaborative
study is a valid basis for the evaluation of measurement uncertainty since, per definition, uncertainty
characterizes the dispersion of the values which may be reasonably attributed to the parameter.
The calculated expanded uncertainty should be ≤  ±2 reproducibility standard deviation.

12.7 Test report

The test report shall specify the following:
— all information necessary for the complete identification of the sample and the date of the test;
— the sampling method used, if known;
— the test method used, with the reference to this document, i.e. ISO 7305:2019;
— all operating details not specified in this document, or regarded as optional, together with details of
any incidents which may have influenced the test result(s).

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Annex A
(informative)

Results of interlaboratory tests

The repeatability, reproducibility and critical differences of the method were determined by means of
two interlaboratory tests organized by BIPEA (Bureau Inter Professionnel d'Etudes Analytiques).
There were 24 laboratories that participated in these tests. Five products were analysed. The statistical
results of the study, assessed in accordance with ISO 5725-2:1994, ISO 5725-3:1994 and ISO 5725-6:1994
are given in Tables A.1 and A.2.

Table A.1 — Statistical results

Durum
Wheat Wheat Wheat Wheat
Sample wheat
semolina flour A flour flour B
semolina
Test No. 1 Test No. 1 Test No. 2 Test No. 1 Test No. 2
Number of laboratories retained after eliminat- 19 20 21 20 21
ing outliers
Mean fat aciditya 12,3 21,0 31,9 52,3 32,7
Repeatability standard deviation, sra 0,57 1,06 0,82 1,06 1,23
Coefficient of variation of repeatability, % 4,7 5,0 2,6 2,0 3,8
Repeatability limit, r (2,77 sr)a 1,58 2,94 2,27 2,94 3,40
Reproducibility standard deviation, sRa 3,43 3,02 4,82 5,23 4,09
Coefficient of variation of reproducibility, % 28,0 14,4 15,1 10,0 12,5
Reproducibility limit, R (2,77 sR)a 9,50 8,37 13,35 14,48 11,32
a Expressed in milligrams of sodium hydroxide per 100 g of dry matter.

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Key
X mean fat acidity
Y standard deviation
Sr
SR

Figure A.1 — Correlation of standard deviations and mean fat acidity level

Table A.2 — Proposed precision table based on Table A.1 and Figure A.1 obtained from the
interlaboratory tests results
mg NaOH sr r sR R CDr CDR
12,3 0,8 2,2 3,2 8,8 1,5 8,4
13,0 0,8 2,2 3,2 8,9 1,6 8,5
13,5 0,8 2,2 3,2 9,0 1,6 8,6
14,5 0,8 2,2 3,3 9,1 1,6 8,7
20,5 0,9 2,4 3,6 10,0 1,7 9,6
30,5 1,0 2,6 4,1 11,5 1,9 11,0
40,5 1,1 2,9 4,7 13,0 2,1 12,5
51,5 1,2 3,2 5,3 14,6 2,3 14,1
52,3 1,3 3,2 5,3 14,7 2,3 14,2

with
sr = 0,009 7x + 0,658 7

sR = 0,053 2x + 2,520 8

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Bibliography

[1] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results —
Part 2: Basic method for the determination of repeatability and reproducibility of a standard
measurement method
[2] ISO 5725-3:1994, Accuracy (trueness and precision) of measurement methods and results — Part 3:
Intermediate measures of the precision of a standard measurement method
[3] ISO 5725-6:1994, Accuracy (trueness and precision) of measurement methods and results — Part 6:
Use in practice of accuracy values

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