Animal feeding stuffs - Enzymatic determination of total starch content (ISO 15914:2004)

ISO 15914:2004 specifies a method for the enzymatic determination of the total starch content of animal feeding stuffs and raw materials for animal feeding stuffs.
The method is also applicable to the determination of starch in starch.
It is important that in the sample matrix no components are present which contribute to the measured extinction at 340 nm.
The analytical range of the method is 40 g/kg to 1 000 g/kg starch. The standard procedure is applicable to the range 200 g/kg to 1000 g/kg. For the lower range, 40 g/kg to 200 g/kg, another dilution procedure for the standard glucose solution and samples can be used.

Futtermittel - Enzymatische Bestimmung von Stärke (ISO 15914:2004)

Diese Internationale Norm legt ein Verfahren zur enzymatischen Bestimmung des Gesamtstärkegehalts in Futtermitteln und Rohmaterialien für Futtermittel fest.
Dieses Verfahren ist auch zur Reinheitsbestimmung der Stärke anwendbar.
Es ist wichtig, dass in der Probenmatrix keine Bestandteile enthalten sind, die bei der Absorption, die bei 340 nm gemessen wird, erfasst werden.
Der Messbereich des Verfahrens liegt bei 40 g/kg bis 1 000 g/kg Stärke. Das Standardverfahren ist anwendbar auf den Bereich von 200 g/kg bis 1 000 g/kg. Für den niedrigeren Bereich von 40 g/kg bis 200 g/kg kann ein anderes Auflösungsverfahren für die Glucosestandardlösung und Proben angewendet werden.

Aliments des animaux - Détermination enzymatique de la teneur totale en amidon (ISO 15914:2004)

Krma - Encimsko določevanje celotnega škroba (ISO 15914:2004)

General Information

Status
Published
Publication Date
31-Aug-2005
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
01-Sep-2005
Due Date
01-Sep-2005
Completion Date
01-Sep-2005

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SLOVENSKI STANDARD
SIST EN ISO 15914:2005
01-september-2005
.UPD(QFLPVNRGRORþHYDQMHFHORWQHJDãNURED ,62

Animal feeding stuffs - Enzymatic determination of total starch content (ISO 15914:2004)

Futtermittel - Enzymatische Bestimmung von Stärke (ISO 15914:2004)

Aliments des animaux - Détermination enzymatique de la teneur totale en amidon (ISO

15914:2004)
Ta slovenski standard je istoveten z: EN ISO 15914:2005
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN ISO 15914:2005 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 15914:2005
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SIST EN ISO 15914:2005
EUROPEAN STANDARD
EN ISO 15914
NORME EUROPÉENNE
EUROPÄISCHE NORM
March 2005
ICS 65.120
English version
Animal feeding stuffs - Enzymatic determination of total starch
content (ISO 15914:2004)

Aliments des animaux - Détermination enzymatique de la Futtermittel - Enzymatische Bestimmung von Stärke (ISO

teneur totale en amidon (ISO 15914:2004) 15914:2004)
This European Standard was approved by CEN on 7 March 2005.

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 Central Secretariat 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 Central Secretariat has the same status as the official

versions.

CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,

Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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

© 2005 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 15914:2005: E

worldwide for CEN national Members.
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SIST EN ISO 15914:2005
EN ISO 15914:2005 (E)
Foreword

The text of ISO 15914:2004 has been prepared by Technical Committee ISO/TC 34 "Agricultural

food products" of the International Organization for Standardization (ISO) and has been taken

over as EN ISO 15914:2005 by Technical Committee CEN/TC 327 "Animal feeding stuffs -

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 September 2005, and conflicting national

standards shall be withdrawn at the latest by September 2005.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of

the following countries are bound to implement this European Standard: Austria, Belgium,

Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,

Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,

Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Endorsement notice

The text of ISO 15914:2004 has been approved by CEN as EN ISO 15914:2005 without any

modifications.
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SIST EN ISO 15914:2005
INTERNATIONAL ISO
STANDARD 15914
First edition
2004-02-01
Animal feeding stuffs — Enzymatic
determination of total starch content
Aliments des animaux — Détermination enzymatique de la teneur totale
en amidon
Reference number
ISO 15914:2004(E)
ISO 2004
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SIST EN ISO 15914:2005
ISO 15914:2004(E)
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ii © ISO 2004 – All rights reserved
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SIST EN ISO 15914:2005
ISO 15914:2004(E)
Contents Page

Foreword............................................................................................................................................................ iv

1 Scope...................................................................................................................................................... 1

2 Normative references ........................................................................................................................... 1

3 Terms and definitions........................................................................................................................... 1

4 Principle ................................................................................................................................................. 2

5 Reagents ................................................................................................................................................ 2

6 Apparatus............................................................................................................................................... 4

7 Preparation of test sample................................................................................................................... 5

8 Procedure............................................................................................................................................... 5

9 Calculation and expression of results ................................................................................................ 7

10 Precision ................................................................................................................................................ 8

11 Test report.............................................................................................................................................. 9

Annex A (informative) Notes on procedure ................................................................................................... 10

Annex B (informative) Results of interlaboratory test .................................................................................. 11

Bibliography ..................................................................................................................................................... 12

© ISO 2004 – All rights reserved iii
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SIST EN ISO 15914:2005
ISO 15914:2004(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies

(ISO member bodies). The work of preparing International Standards is normally carried out through ISO

technical committees. Each member body interested in a subject for which a technical committee has been

established has the right to be represented on that committee. International organizations, governmental and

non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the

International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 15914 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal

feeding stuffs.
iv © ISO 2004 – All rights reserved
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SIST EN ISO 15914:2005
INTERNATIONAL STANDARD ISO 15914:2004(E)
Animal feeding stuffs — Enzymatic determination of total starch
content
1 Scope

This international Standard specifies a method for the enzymatic determination of the total starch content of

animal feeding stuffs and raw materials for animal feeding stuffs.
The method is also applicable to the determination of the purity of starch.

It is important that in the sample matrix no components are present which contribute to the measured

extinction at 340 nm.

The analytical range of the method is 40 g/kg to 1 000 g/kg starch. The standard procedure is applicable to

the range 200 g/kg to 1000 g/kg. For the lower range, 40 g/kg to 200 g/kg, another dilution procedure for the

standard glucose solution and samples can be used.
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 3696:1987, Water for analytical laboratory use — Specification and test methods

ISO 6498:1998, Animal feeding stuffs — Preparation of test samples
3 Terms and definitions
For the purpose of this document, the following terms and definitions apply.
3.1
starch

natural vegetable polymer consisting of long linear unbranched chains of 1,4-α-D-glucose units (amylose)

and/or long α-1,6-branched chains of α-1,4-linked glucose units (amylopectin)
3.2
starch content

mass fraction of starch and its high molecular mass breakdown products, insoluble in 40 % ethanol, and

determined in accordance with the method of this International Standard
NOTE The starch content is expressed in grams per kilogram.
© ISO 2004 – All rights reserved 1
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SIST EN ISO 15914:2005
ISO 15914:2004(E)
4 Principle

The milled test sample is extracted with 40 % ethanol to remove the soluble sugars. Sample disintegration

and starch solubilization is achieved by dispersing the extracted test portion in aqueous DMSO (90 % volume

fraction) at 100 °C, followed by addition of concentrated hydrochloric acid at 60 °C. The solubilized and

dissolved starch is quantitatively converted into glucose by the enzyme amyloglucosidase. The glucose

quantification is carried out by the well-known hexokinase method (see [1], [2]).

5 Reagents
Use only reagents of recognized analytical grade.
5.1 Water, complying with at least grade 3 in accordance with ISO 3696:1987.
5.2 Ethanol (C H OH), 40 % (volume fraction)
2 5
Take 417 ml of ethanol (96 % volume fraction) and dilute with water to 1 000 ml
5.3 Hydrochloric acid, c(HCl) = 12 mol/l.
5.4 Aqueous sodium hydroxide, c(NaOH) = 4 mol/l.

Dissolve in a beaker 40 g of NaOH in about 50 ml water. After cooling, transfer quantitatively to a 250 ml

volumetric flask and dilute to the mark with water.
WARNING — Heat develops. Wear safety glasses.
5.5 Acetic acid solution, c(CH COOH) = 2 mol/l.

Add to a 500 ml volumetric flask about 200 ml water, followed by 59 ml of glacial acetic acid. Dilute to the

mark with water.
5.6 Sodium acetate solution, c(CH COONa) = 2 mol/l.

Dissolve, in a 500 ml volumetric flask, 82,0 g of sodium acetate in water and dilute to the mark with water.

5.7 Sodium acetate buffer, c(CH COONa/H) = 2 mol/l, pH = 4,8.

Mix 41 ml of acetic acid solution (5.5) with 59 ml of sodium acetate solution (5.6). Check the pH with a

pH-meter and, if necessary, adjust to obtain the correct pH with the acetic acid or the sodium acetate solution.

Prepare a fresh buffer solution daily.
5.8 Aqueous dimethylsulfoxide, (σ ) = 90 % (volume fraction).
DMSO
Mix pure DMSO and water in a volume ratio of 9:1.
5.9 Clarifying solutions, according to Carrez, prepared as follows.
5.9.1 Potassium hexacyanoferrate(II) solution, c[K Fe(CN) ] = 0,25 mol/l.
4 6

In a 1 litre volumetric flask, dissolve 106 g of potassium hexacyanoferrate(II) trihydrate [K Fe(CN) ⋅3H O] in

4 6 2
water. Dilute to the mark with water.
5.9.2 Zinc acetate, solution in 0,5 mol/l acetic acid, c[Zn(CH CO ) ] = 1 mol/l.
3 2 2
2 © ISO 2004 – All rights reserved
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SIST EN ISO 15914:2005
ISO 15914:2004(E)

In a 1 litre volumetric flask, dissolve 219,5 g of zinc acetate dihydrate [Zn(CH CO ) ⋅2H O] and 30 g of glacial

3 2 2 2
acetic acid in water. Dilute to the mark with water.
5.10 Iodine solution in potassium iodide

In a 1 litre volumetric flask, dissolve 12,7 g of iodine (I ) and 24,0 g of potassium iodide (KI) in water. Dilute to

the mark with water. Dilute this solution 10-fold before use.
5.11 Standard glucose solution
5.11.1 Samples containing 200 g/kg to 1 000 g/kg starch

Prepare three independent standard glucose solutions (c = 0,0194 mol/l). In each 100 ml volumetric flask

dissolve 350 mg anhydrous glucose (C H O ) to the nearest mg in water. Dilute to the mark with water.

6 12 6
5.11.2 Samples containing 40 g/kg to 200 g/kg starch

Prepare three independent standard glucose solutions (c = 0,0039 mol/l). In each 500 ml volumetric flask,

dissolve 350 mg ± 1 mg of anhydrous glucose (C H O ) in water. Dilute to the mark with water.

6 12 6
Prepare fresh standard glucose solutions daily.
5.12 Amyloglucosidase solution, 160 U/ml AMG.

Dissolve, in a mixture of 9 ml of water and 1 ml of sodium acetate buffer (5.7), 267 mg of amyloglucosidase

(AMG) [EC 3.2.1.3 (Aspergillus niger; Roche Diagnostics, No. 1 202 367, 6 U/mg)]. With respect to storage

and the use of the enzymes, follow carefully the recommendations of the enzyme supplier.

When another enzyme is used, the activity should be estimated as given in Note 1. The mass of the enzyme

should be adapted to the activity found.

NOTE 1 Different enzyme suppliers use different definitions for the units of activities of enzymes. In this International

Standard the following definition for the unit of activity for AMG has been used: 1 unit of amyloglucosidase will release

1 µmol of glucose from glycogen in 1 min at 25 °C and pH = 4,75.
NOTE 2 10 ml of enzyme solutions is enough for about 75 determinations.

5.13 D-Glucose UV test set, for quantifying glucose enzymaticly, with the hexokinase method (R-Biopharm,

No. 716251 , according to the manufacturer, the unused kits may be stored for 1 year at 4 °C), as given in

5.13.1 to 5.13.3.
5.13.1 Buffer/substrate solution (Bottle 1)

Dissolve the content of Bottle 1 in 45 ml of freshly prepared distilled water. Store this reagent in a cool (4 °C)

and dark place for not longer than for 4 weeks. Use the required amount of this solution at ambient

temperature.
5.13.2 Enzyme solution (Bottle 2)
This solution is ready for use.

1) Roche Diagnostics No. 1 202 367 is an example of a suitable product available commercially. This information is

given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this

product.

2) R-Biopharm AG No. 716251 is an example of a suitable product available commercially. This information is given for

the convenience of users of this International Standard and does not constitute an endorsement by ISO of this products.

© ISO 2004 – All rights reserved 3
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SIST EN ISO 15914:2005
ISO 15914:2004(E)
5.13.3 Colouring solution

Mix 22,5 ml of buffer/substrate solution (5.13.1) with 95 ml of water and 0,45 ml of enzyme solution (5.13.2)

and homogenize. This amount of reagent is enough for a series of about 40 measurements. If more or less

measurements are be carried out, the mixing volumes may be adapted.
Only use fresh prepared colouring solutions at ambient temperature.
6 Apparatu
...

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