ISO 13965:1998

Meat and meat products — Determination of starch and glucose contents — Enzymatic method

Viande et produits à base de viande — Détermination des teneurs en amidon et en glucose — Méthode enzymatique

General Information

• Status: Published
• Publication Date: 16-Sep-1998
• ICS: 67.120.10 - Meat and meat products
• Technical Committee: ISO/TC 34/SC 6 - Meat, poultry, fish, eggs and their products
• Drafting Committee: ISO/TC 34/SC 6 - Meat, poultry, fish, eggs and their products
• Current Stage: 9093 - International Standard confirmed
• Completion Date: 10-Aug-2020

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 13965
First edition
1998-09-15
Meat and meat products — Determination
of starch and glucose contents —
Enzymatic method
Viande et produits à base de viande — Détermination des teneurs en
amidon et en glucose — Méthode enzymatique
A
Reference number
ISO 13965:1998(E)

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ISO 13965:1998(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.
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 13965 was prepared by Technical Committee
ISO/TC 34, Agricultural food products, Subcommittee SC 6, Meat and meat
products.
Annexes A and B of this International Standard are for information only.
©  ISO 1998
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 the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet [e-mail]
Printed in Switzerland
ii

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INTERNATIONAL STANDARD  ISO ISO 13965:1998(E)
Meat and meat products — Determination of starch and
glucose contents — Enzymatic method
1 Scope
This International Standard specifies an enzymatic method for the determination of water-free starch content and
glucose content of all kinds of meat and meat products, including poultry.
The method is suitable for the quantitative determination of starch and glucose contents down to levels of
0,30 % (m/m).
The method is not applicable for chemically modified starches or their derivatives.
2 Normative reference
The following standard contains provisions which, through reference in this text, constitute provisions of this
International Standard. At the time of publication, the edition indicated was valid. All standards are subject to
revision, and parties to agreements based on this International Standard are encouraged to investigate the
possibility of applying the most recent edition of the standard indicated below. Members of IEC and ISO maintain
registers of currently valid International Standards.
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods.
3 Definitions
For the purposes of this International Standard, the following definitions apply.
3.1
starch content of meat and meat products
starch content determined in accordance with the procedure described in this International Standard, and
expressed as a percentage by mass
3.2
glucose content of meat and meat products
glucose content determined in accordance with the procedure described in this International Standard, and
expressed as a percentage by mass
4 Principle
4.1 Hydrolysis of the starch present in a test portion with the enzyme a-amylase at pH = 5,0 for 15 min.
Determination of the starch content using the following enzymatic reactions.
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ISO 13965:1998(E)
4.2 Hydrolysis of the solubilized starch to yield glucose using amyloglucosidase (AGS):
starch + (n21)H O glucose
2
For the determination of the glucose content, this step is omitted.
4.3 Phosphorylation of the glucose generated by means of adenosine 5'-triphosphate (ATP) to yield glucose
6-phosphate (G-6-P) using hexokinase (HK):
glucose + ATP glucose 6-phosphate + ADP
4.4 Oxidation of glucose 6-phosphate (G-6-P) by means of nicotinamide adenine dinucleotide phosphate
(NADP) to gluconate 6-phosphate using glucose 6-phosphate dehydrogenase (G-6-PDH):
+ +
glucose 6-phosphate + NADP gluconate 6-phosphate + NADPH + H
4.5 Spectrometric measurement of the amount of reduced nicotinamide dinucleotide phosphate (NADPH) at a
wavelength of 340 nm.
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
5.1 Water, complying with at least grade 3 in accordance with ISO 3696.
5.2 a-Amylase (EC 3.2.1.1), enzyme suspension.
1)
A liquid enzyme preparation of a heat-stable a-amylase produced from Bacillus licheniformis (Termamyl® 120L) .
5.3 Sodium hydroxide solution, c(NaOH) = 5 mol/l.
Dissolve 200 g of sodium hydroxide in water. Cool to room temperature, dilute to 1000 ml and mix.
5.4 Sodium hydroxide solution, c(NaOH) = 0,5 mol/l.
Dissolve 20 g of sodium hydroxide in water. Cool to room temperature, dilute to 1000 ml and mix.
5.5 Ammonium sulfate solution, c[(NH ) SO ] = 3,2 mol/l.
4 2 4
Dissolve 422 g of ammonium sulfate in water. Dilute to 1000 ml and mix.
5.6 Acetate buffer, c(CH CO Na) = 0,1 mol/l, pH = 5,0.
3 2
Dissolve 6,80 g of sodium acetate trihydrate (CH CO Na{3H O) in 400 ml of water. Adjust the pH to 5,0 with
3 2 2
hydrochloric acid or sodium hydroxide solution with a pH-meter (6.2). Dilute with water to 500 ml and mix.
The solution is stable for at least 3 months at +4 °C in the dark.
5.7 Citrate buffer, c(citrate) = 0,05 mol/l, pH = 4,6.
Dissolve 440 mg of citric acid monohydrate (CHO{HO) and 850 mg of trisodium citrate dihydrate
6 8 7 2
(CHNaO{2H O) in water. Dilute with water to 100 ml and mix. Check the pH with a pH-meter (6.2) and adjust if
6 5 3 7 2
necessary with hydrochloric acid or sodium hydroxide solution.
The solution is stable for at least 3 months at +4 °C in the dark.

1)  Termamyl® 120 L is an example of a suitable product available commercially from Novo, Denmark, and Tecator, Sweden.
This information is given for the convenience of users of this International Standard and does not constitute an endorsement by
ISO of this product.
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ISO 13965:1998(E)
5.8 Triethanolamine buffer, c(triethanolamine) = 0,75 mol/l, pH = 7,6.
Dissolve 14,0 g of triethanolamine hydrochloride (C H NO {HCl) and 0,25 g of magnesium sulfate heptahydrate
6 15 3
(MgSO {7H O) in 80 ml of water. Set the pH at 7,6 with a pH-meter, using the sodium hydroxide solutions (5.3 and
4 2
5.4). Dilute with water to 100 ml and mix.
The solution is stable for 4 weeks at +4 °C in the dark.
−3
(b-NADP-Na ) = 12,7 x 10 mol/l.
5.9 Nicotinamide adenine dinucleotide phosphate solution, c
2
Dissolve 100 mg of NADP disodium salt in 10,0 ml of water and mix.
The solution is stable for 4 weeks at +4 °C in the dark.
−3
5.10 Adenosine-5'-triphosphate solution, c(5'-ATP-Na H {3H O) ' 81 x 10 mol/l.
2 2 2
Dissolve 500 mg of 5'-ATP-Na H {3H O and 500 mg of anhydrous monosodium hydrogen carbonate (NaHCO ) in
2 2 2 3
10,0 ml of water and mix.
The solution is stable for 4 weeks at +4 °C in the dark.
5.11 Amyloglucosidase (AGS; EC 3.2.1.3), enzyme suspension in ammonium sulfate solution (5.5),
r(AGS) = 10 mg/ml.
The specific activity of the enzyme shall be 14 units per milligram.
The suspension is stable for 1 year at +4 °C in the dark.
5.12 Hexokinase (HK; EC 2.7.1.1)/glucose 6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49), enzyme
suspension in ammonium sulfate solution (5.5), r(HK) = 2 mg/ml and r(G-6-PDH) = 1 mg/ml.
The specific activity of both enzymes shall be 140 units per milligram.
The suspension is stable for 1 year at +4 °C in the dark.
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 Mechanical or electrical equipment, capable of homogenizing the laboratory sample.
This includes a high-speed rotational cutter, or a mincer fitted with a plate with apertures not exceeding 4,0 mm in
diameter.
6.2 pH-meter.
6.3 Fluted filter papers, glucose free, of diameter about 15 cm.
6.4 Pipettes, calibrated, for enzymatic analysis, or automatic micropipettes of equivalent quality with the
following volumes: 20 ml, 50 ml, 100 ml and 200 ml.
6.5 Small plastics spatula, for mixing the contents of a cuvette (6.9).
6.6 Water bath, capable of being maintained at (60 ± 2) °C.
6.7 Hot plate.
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6.8 Spectrometer, capable of measuring at a wavelength of 340 nm.
NOTE When a spectrometer fitted with a mercury vapour lamp is available, the readings can be carried out at 365 nm and
−1 −1 −1 −1
334 nm. The molecular absorption coefficient k for NADPH is 3,51 l{mmol {cm at 365 nm and 6,18 l{mmol {cm at 334 nm.
6.9 Cuvettes, made of quartz or glass, with lid, or disposable cuvettes for single use, made of polymethacrylate,
of 10 mm optical path length.
6.10 Analytical balance, capable of weighing to the nearest 0,1 mg.
7 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling method is
given in ISO 3100-1 [1].
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changed during transport or storage.
Start from a representative sample of at least 200 g. Store the sample in such a way that deterioration and change
in composition are prevented.
8 Preparation of test sample
Homogenize the laboratory sample with the appropriate equipment (6.1). Take care that the temperature of the
sample material does not rise above 25 °C. If a mincer is used, pass the sample at least twice through the
equipment.
Fill a suitable airtight container with the prepared sample. Close the container and store in such a way that
deterioration and change in composition are prevented. Analyse the sample as soon as practicable, but always
within 24 h after homogenization.
9 Procedure
NOTE Muscle glycogen, which occurs normally in, for example, sausages, does not interfere with the determination. Maltose
interferes because this disaccharide is hydrolysed by amyloglucosidase into glucose. Maltose (and glucose) can, however, be
extracted from the sample with alcohol.
9.1 Test portion
If the test sample contains maltose, make sure that the water content does not exceed 20 % (m/m). If necessary, dry
the test sample.
Weigh about 400 mg of the prepared test sample (see clause 8) to the nearest 0,1 mg into a centrifuge tube.
Proceed in accordance with 9.2.
If the test sample does not contain maltose, weigh between 100 mg and 1,0 g (m) of the prepared test sample to the
nearest 0,1 mg into a 100 ml conical flask.
Add 30 ml of acetate buffer (5.6) and proceed in accordance with 9.3.
9.2 Extraction of maltose (and glucose)
Wash the sample three times with 10 ml of 40 % (V/V) ethanol and centrifuge after each washing. Filter the
supernatant. Combine the precipitate in the centrifuge tube with that on the filter and transfer to a 100 ml conical
flask using 4 x 5 ml of acetate buffer (5.6). Add 10 ml of acetate buffer (5.6).
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9.3 Preparation of extract
Pipette (6.4) 50 ml of a-amylase su
...