SIST EN ISO 15914:2005

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
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 01-Sep-2005
• Due Date: 01-Sep-2005
• Completion Date: 01-Sep-2005
• Directive: 70/373/EC - Council Directive 70/373/EEC of 20 July 1970 on the introduction of Community methods of sampling and analysis for the official control of feeding-stuffs
• Directive: 96/25/EC - Council Directive 96/25/EC of 29 April 1996 on the circulation of feed materials, amending Directives 70/524/EEC, 74/63/EEC, 82/471/EEC and 93/74/EEC and repealing Directive 77/101/EEC
• Mandate: M/315 - Mandate to CEN for standardization in the field of methods of analysis for animal feedingstuffs
• Ref Project: EN ISO 15914:2005 - Animal feeding stuffs - Enzymatic determination of total starch content (ISO 15914:2004)

Overview

EN ISO 15914:2005 (ISO 15914:2004) specifies an enzymatic laboratory method for the determination of total starch content in animal feeding stuffs and raw materials. Adopted by CEN, the method is also applicable to assessing the purity of starch. The procedure quantifies starch after solubilization and enzymatic conversion to glucose, followed by glucose measurement using the hexokinase spectrophotometric method (measurement at 340 nm).

Key facts:

• Analytical range: 40 g/kg to 1 000 g/kg starch
• Standard procedure applicable: 200 g/kg to 1 000 g/kg
• Alternate dilution procedure provided for 40 g/kg to 200 g/kg
• Important requirement: sample matrix must not contain components that absorb at 340 nm

Key topics and technical requirements

• Principle: Remove soluble sugars with 40% ethanol, solubilize starch in aqueous DMSO (90% v/v) at 100 °C and partially hydrolyse with concentrated HCl at 60 °C. Convert solubilized starch to glucose using amyloglucosidase; quantify glucose by the hexokinase enzymatic assay with spectrometric detection at 340 nm.
• Reagents: 40% ethanol, DMSO (90%), HCl (12 mol/L), amyloglucosidase solution (activity-specified), standard glucose solutions (different concentrations for two analytical ranges), Carrez clarifying reagents, acetate buffers.
• Apparatus: calibrated analytical balance, centrifuge, thermostatted water baths (100 °C and 60 °C), pH metre, pipettes, spectrophotometer capable of measuring at 340 nm, and appropriate centrifuge/ reaction tubes.
• Sample preparation: Follow ISO 6498 for test sample preparation; extraction removes soluble sugars prior to starch solubilization.
• Precision and reporting: The standard includes calculation, expression of results, precision data and reporting requirements for reliable inter-laboratory comparability.

Practical applications and users

EN ISO 15914:2005 is designed for laboratories and organizations involved in:

• Feed quality control and formulation (feed manufacturers, nutritionists)
• Raw material suppliers and starch producers (purity checks)
• Contract analytical laboratories and university research labs analyzing feed composition
• Regulatory and compliance testing for animal feeding stuffs

Benefits:

• Provides a validated, enzyme-based approach that targets total starch (including high-molecular-weight breakdown products)
• Enables consistent, comparable results across laboratories when following specified reagents, enzyme activities and analytical ranges

Related standards

• ISO 6498 - Preparation of test samples for animal feeding stuffs (referenced for sample prep)
• ISO 3696 - Water for analytical laboratory use (quality of water required)

Keywords: EN ISO 15914:2005, ISO 15914:2004, animal feeding stuffs, enzymatic determination, total starch content, starch analysis, amyloglucosidase, hexokinase method, 340 nm, DMSO, ethanol extraction.

Standards Content (Sample)

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

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.

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.
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
ISO 15914:2004(E)
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ii © ISO 2004 – All rights reserved

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 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

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 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

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
1)
(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,
2)
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 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
...