Modified starch - Determination of acetyl content - Enzymatic method (ISO 11213:1995)

Applies to modified starch, both granular and soluble in cold water. The methods specified enable to measure total and free acetyl contents; bound acetyl content is calculated as the difference of the two values. Total content is determined by heating the sample with dilute hydrochloric acid and converting the acetate with adenosine-5-triphosphate (ATP) and coenzyme A (CoA) to acetyl-CoA. The methods are suitable for determining acetyl contents up to 2 % (m/m).

Modifizierte Stärke - Bestimmung des Acetylgehaltes - Enzymatisches Verfahren (ISO 11213:1995)

Diese Internationale Norm legt ein enzymatisches Verfahren zur Bestimmung des Acetylgehaltes von modifizierter Stärke fest, die sowohl granuliert als auch kaltwasserlöslich ist. Der Gesamtacetylgehalt und der Gehalt an freiem Acetyl werden bestimmt und der Gehalt an gebundenem Acetyl wird berechnet. Das Verfahren ist zur Bestimmung von Acetylgehalten bis zu 2 % (m/m) anwendbar.

Amidon modifié - Dosage de l'acétyle - Méthode enzymatique (ISO 11213:1995)

La présente Norme internationale prescrit une méthode enzymatique pour le dosage de l'acétyle contenu dans l'amidon modifié, granulaire ou soluble dans l'eau froide. Les teneurs en acétyle total et libre sont déterminées et la teneur en acétyle lié est calculée. La méthode convient pour déterminer des teneurs en acétyle jusqu'à 2 % (m/m).

Modificiran škrob - Določevanje acetilnih skupin - Encimatska metoda (ISO 11213:1995)

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SIST EN ISO 11213:1998

Modified starch - Determination of acetyl content - Enzymatic method (ISO 11213:1995)
Modifizierte Stärke - Bestimmung des Acetylgehaltes - Enzymatisches Verfahren (ISO
Amidon modifié - Dosage de l'acétyle - Méthode enzymatique (ISO 11213:1995)
Ta slovenski standard je istoveten z: EN ISO 11213:1995
67.180.20 Škrob in izdelki iz njega Starch and derived products
SIST EN ISO 11213:1998 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 11213:1998

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SIST EN ISO 11213:1998

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SIST EN ISO 11213:1998

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SIST EN ISO 11213:1998

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SIST EN ISO 11213:1998

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SIST EN ISO 11213:1998
First edition
Modified starch - Determination of acetyl
content - Enzymatic method
Amidon modifie - Dosage de I ’acbtye - M6 thode enzyma tique
Reference nmber
ISO 11213:1995(E)

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SIST EN ISO 11213:1998
ISO 11213:1995(E)
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. Esch 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.
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.
International Standard ISO 11213 was prepared by Technical Committee
lSO/TC 93, Starch (including derivatives and by-products).
Annexes A, B and C of this International Standard are for information only.
0 ISO 1995
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced
or utilized in any form or by any means, electronie or mechanrcal, rncluding photocopying and
mrcrofilm, without permisston In writing from the publisher.
lnternatronal Organizatron for Standardization
Case Postale 56 l CH-l 211 Geneve 20 l Switzerland
Printed in Switzerland

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SIST EN ISO 11213:1998
Modified starch - Determination of acetyl content -
Enzymatic method
then reacts with oxaloacetate to form citrate in the
1 Scope
presence of citrate synthase (CS).
This International Standard specifies an enzymatic
The oxaloacetate required for the reaction is formed
method for the determination of the acetyl content
from malate and nicotinamide adenine dinucleotide
of modified starch, both granular and soluble in cold
(NAD) in the presence of malate-dehydrogenase
water. Total and free acetyl contents are determined
(MDH). In this reaction, the NAD is reduced to NADH
and the bound acetyl content is calculated.
and the formation of NADH tan be determined by
measuring the increase in absorbance at a specified
The method is suitable for determining acetyl con-
wavelength. (See reference [1] in annex C.)
tents up to 2 % (m/m).
The free acetyl content is determined by making a
2 Normative references
Suspension of the modified starch in water, filtering,
and determining the acetyl content of the filtrate as
The following Standards contain provisions which,
already described. The bound acetyl content is calcu-
through reference in this text, constitute provisions
lated by subtracting the free acetyl content from total
of this International Standard. At the time of publi-
acetyl content.
cation, the editions indicated were valid. All Standards
are subject to revision, and Parties to agreements
4 Reagents and materials
based on this International Standard are encouraged
to investigate the possibility of applying the most re-
The reagents used shall be of recognized analytical
cent editions of the Standards indicated below.
grade, unless otherwise specified. The water used
Members of IEC and ISO maintain registers of cur-
shall comply with the specifications of ISO 3696,
rently valid International Standards.
grade 2. The enzymes used shall be of a quality
equivalent to the relevant enzymes of Boehringer
ISO 1666: 1973, Starch - Determination of moisture
content - Oven-drying methods.
NOTE 1 Suitable test kits which are commercially avail-
ISO 3696: 1987, Water for analytical laboratory use -
able tan be used.
Specification and test methods.
4.1 Hydrochlorit acid, 1 mol/1 solution.
3 Principle
4.2 Sodium hydroxide, 5 mol/1 Solution.
The total acetyl content is determined by heating the
Sample with dilute hydrochloric acid which hydrolyses
the acetyl fraction and solubilizes the starch. In the
4.3 Buffer Solution.
presence of the enzyme acetyl-CoA synthetase (ACS),
In about 70 ml of water, dissolve the following re-
acetate is converted with adenosine-5-triphosphate
(ATP) and coenzyme A (CoA) to acetyl-Co-A. The latter
1) This information is given for the convenience of users of this International Standard and does no ’t constitute an endorsement
by ISO of the products named.

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SIST EN ISO 11213:1998
ISO 11213:1995(E)
7,5 g of triethanolamine;
5 Apparatus
420 mg of L-malic acid;
Usual laboratory apparatus and in particular the fol-
210 mg of magnesium chloride hexahydrate
(MgCI,.GH,O) .
5.1 Conical flasks, of capacity 250 ml, equipped
with screw taps.
Add as much potassium hydroxide 5 mol/1 Solution as
necessary in Order to obtain a pH of 8,4. The volume
Boiling water bath, equipped with a shaker.
required is about 8 ml.
5.3 Volumetric flasks, of capacity 200 ml.
The Solution is stable for 1 year when at
+ 4 “C.
5.4 Micropipettes or syringes.
5.5 Molecular absorption spectrometer, suita ble
4.4 ATB-CoA-NAD Solution.
for Operation at 340 nm.
In 20 ml of water, dissolve the following reagents:
5.6 Cuvettes, of quartz glass or other materials
at 340 nm, with a thickness of
500 mg of crystallized disodium salt trihydrate of transparent
adenosine-5 ’-triphosphate [ATP-Na,H,.3H,O; IO mm + 0,l mm.
98 % (dm)];
5.9 Sieve, with an aperture of 800 Pm.
500 mg of anhydrous sodium hydrogen carbonate;
5.8 Blade mill.
50 mg of lyophilized trilithium salt of coenzyme A
[about 85 % (m/m) CoA];
5.9 Water bath, capable of being thermostatically
controlled between 20 “C and 25 “C.
250 mg of lyophilized free acid monohydrate of
nicotinamide adenine dinucleotide
6 Preparation of the Sample
[--NAD.H,O > 98 % (m/m)].
Sieve through a 800 Pm sieve (5.7). If material does
The Solution is stable for 1 week when stored at
not pass through the sieve, grind the Sample with a
+ 4 “C.
blade mill (5.8) so that it will completely pass through
the 800 Pm sieve. Homogenize the Sample.
4.5 MDH-CS Suspension .
7 Procedure
Disperse about 1 100 U (international units)
malate-dehydrogenase (MDH from pig heart;
7,l Hydrolysis of acetyl groups
EC 1 .l .1.37) and about 270 U of citrate synthase (CS
from pig heart; EC 4.1.37) in 0,4 ml of ammonium
7.1.1 Dispersion of granular starch
sulfate Solution, c[(NH&SOJ = 3,2 mol/l.
Weigh, to the nearest 1 mg, approximately 1 g of the
The Solution is sta ble for 1 year when stored at
prepared Sample and place it in a conical flask (5.1).
+ 4 “C.
Add 50 ml of hydrochloric acid (4.1) while agitating to
NOTE 2 One international unit catalyses
ensure good dispersion. Continue as described in
(1 u>
1 pmol/min at 25 “C from the relevant Substrate.
7.12 Dispersion of pregelatinized starch
4.6 ACS Solution.
Add 50 ml of hydrochloric acid (4.1) to a conical flask
(5.1). Introduce a magnetic stirrer and Start agitation.
Dissolve 20 mg of lyophilizate containing 5 mg of
Slowly and carefully add about 1 g of the prepared
acetyl-coenzyme A synthetase (ACS from yeast;
Sample. Ensure a good, lump-free dispersion. Deter-
EC 6.2.1 .l; z 16 U) in 0,4 ml of water.
mine the mass 0% the test Portion by weighing by
differente to the nearest 1 mg.
The Solution is stable for 5 d when stored at + 4 “C.

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SIST EN ISO 11213:1998
ISO 11213:1995(E)
7.1.3 Hydrolysis and filtration anhydrous sodium acetate. Then transfer to a
1 000 ml volumetric flask. Place the volumetric flask
Seal the conical flask tightly with the screw cap and
in the water bath (5.9) for temperature equilibration
place it in the boiling water bath (5.2) with the shaker
between 20 “C and 25 “C. Control the temperature
operating for 30 min.
and make up to the mark with distilled water. Con-
tinue as described in 7.4.
Remove the flask and cool to about 20 “C + 5 “C by
immersing it in an ice bath. Open the flask when its
7.4 Enzymatic determination of acetic acid
content is fully cooled, add 10 ml of sodium hydroxide
Solution (4.2), mix, and wash the contents quantitat-
Carry out the enzymatic determination of acetic acid
ively into a 200 ml volumetric flask (5.3). Place the
according to the following analytical arrangement and
volumetric flask in the water bath (5.9) for tempera-
ture equilibration between 20 “C and 25 “C. Control
the temperature and make up to the mark with dis-
- wavelength: 340 nm;
tilled water. Filter through a suitable filter Paper. Re-
ject the first 20 ml to 30 ml of filtrate and directly use
- temperature: 20 “C to 25 “C.
the remaining Solution as the test Solution in the
enzymatic determination, as described in 7.4.
Read the absorbances against a cuvette (5.6) filled
with wate

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