ISO 4134:2021

Meat and meat products - Determination of L-(+)-glutamic acid content - Reference method

This document specifies the spectrophotometer method and the light absorption microplate reader method for the determination of the free L-(+)-glutamic acid content of meat and meat products. This document is applicable to meat and meat products, including livestock and poultry products.

Viande et produits à base de viande — Détermination de la teneur en acide L-(+)-glutamique — Méthode de référence

General Information

• Status: Published
• Publication Date: 22-Jul-2021
• 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/WG 25 - Determination of glutamic acid content
• Current Stage: 6060 - International Standard published
• Start Date: 23-Jul-2021
• Due Date: 12-Feb-2022
• Completion Date: 23-Jul-2021

Relations

• Consolidated By: ISO 294-5 - Plastics - Injection moulding of test specimens of thermoplastic materials - Part 5: Preparation of standard specimens for investigating anisotropy
• Effective Date: 03-Dec-2022
• Revises: ISO 4134:1999 - Meat and meat products - Determination of L-(+)- glutamic acid content - Reference method
• Effective Date: 23-Apr-2020

Overview

ISO 4134:2021 - Meat and meat products - Determination of L-(+)-glutamic acid content - Reference method - defines laboratory reference procedures for measuring free L-(+)-glutamic acid (glutamate) in meat and meat products (including livestock and poultry). The standard specifies two validated assay routes: the spectrophotometer method and the light absorption microplate reader method. Both rely on enzymatic conversion of L-(+)-glutamic acid (glutamate dehydrogenase and NAD) and a colorimetric formazan end‑point measured at 490 nm.

Key topics and requirements

• Scope and applicability: Free L-(+)-glutamic acid in fresh and processed meat and poultry products.
• Principle: Extraction of free glutamic acid with perchloric acid, pH adjustment (to alkaline), enzymatic reduction of NAD by glutamate dehydrogenase, NADH-driven reduction of iodonitrotetrazolium chloride via diaphorase to form a formazan dye measured at 490 nm.
• Two validated methods:
  • Spectrophotometer method (detailed reagents, apparatus, extraction and measurement steps).
  • Light absorption microplate reader method (higher throughput option using microplates).
• Reagents and conditions: Includes dilute perchloric acid extraction (c = 1.0 mol/L), triethanolamine phosphate buffer (pH 8.6), KOH solutions for neutralization, and defined enzyme/NAD mixtures.
• Analytical performance: Includes instructions for calibration (standard curves and linear regression), calculation of L-(+)-glutamic acid concentration, and documented precision metrics - repeatability, reproducibility, interlaboratory test results and detection limit.
• Sampling & sample prep: Sampling is not prescriptive but recommends representative sampling (see CAC/GL 50-2004), homogenization, temperature control and storage instructions to avoid composition changes.
• Reporting and safety: Specifies minimum reporting content for test reports and includes an informative annex on safety practices when handling strong acids and reagents.

Applications

• Quality control in meat processing (flavour profiling, glutamate content monitoring).
• Regulatory and compliance testing where free glutamate must be quantified.
• Food research and product development investigating umami contributors or ingredient effects.
• High-throughput laboratories can adopt the microplate reader method for increased sample throughput while maintaining reference-method traceability.

Who should use this standard

• Food testing laboratories, meat processors, regulatory agencies, contract testing providers, and researchers requiring a validated reference method for quantifying free L-(+)-glutamic acid in meat and poultry products.

Related standards

Normative references cited include ISO 648, ISO 1042, ISO 1442 (moisture reference), ISO 3696 (water for analytical use) and ISO 8655‑2 (piston pipettes). These support laboratory equipment, sampling and quality controls referenced in ISO 4134:2021.

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 4134
Third edition
2021-07
Meat and meat products —
Determination of L-(+)-glutamic acid
content — Reference method
Viande et produits à base de viande — Détermination de la teneur en
acide L-(+)-glutamique — Méthode de référence
Reference number
©
ISO 2021
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Sampling . 2
6 Preparation of test sample . 2
7 Test method of spectrophotometer . 2
7.1 Reagents. 2
7.2 Apparatus . 4
7.3 Procedure . 5
7.3.1 General. 5
7.3.2 Test portion . 5
7.3.3 Preparation of extract . . 5
7.3.4 Determination . 5
7.4 Calculation and results . 6
7.4.1 Absorbance difference of L-(+)-glutamic acid standard solution . 6
7.4.2 Absorbance difference of L-(+)-glutamic acid test solution . 7
7.4.3 Formula of linear regression of L-(+)-glutamic acid standard curve . 7
7.4.4 L-(+)-glutamic acid concentration of the test solution . 7
7.4.5 L-(+)-glutamic acid content of test sample . 8
7.5 Precision . 8
7.5.1 Interlaboratory test . 8
7.5.2 Repeatability . 8
7.5.3 Reproducibility . 8
7.6 Detection limit . 8
8 Test method of light absorption microplate reader . 9
8.1 Reagents. 9
8.2 Apparatus . 9
8.3 Procedure . 9
8.3.1 General. 9
8.3.2 Extraction of L-(+)-glutamic acid in the test portion . 9
8.3.3 Determination . 9
8.4 Calculation and results .10
8.4.1 Absorbance difference for L-(+)-glutamic acid standard solution .10
8.4.2 Absorbance difference for L-(+)-glutamic acid test solution.10
8.4.3 Formula of linear regression for L-(+)-glutamic acid standard curve .11
8.4.4 L-(+)-glutamic acid concentration of the test solution .11
8.4.5 L-(+)-glutamic acid content of test sample .11
8.5 Precision .12
8.5.1 Interlaboratory test .12
8.5.2 Repeatability .12
8.5.3 Reproducibility .12
8.6 Detection limit .12
9 Test report .12
Annex A (informative) Safety practices .14
Bibliography .15
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.
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).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
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 6,
Meat, poultry, fish, eggs and their products.
This third edition cancels and replaces the second edition (ISO 4134:1999), which has been technically
revised. The main changes compared with the previous edition are as follows:
— a new test method, the light absorption microplate reader method, has been added;
— the order of the clauses has been rearranged;
— the Scope (Clause 1) has been revised to specify free L-(+)-glutamic acid in meat and meat products;
— the Normative references (Clause 2) have been updated;
— the Terms and definitions (Clause 3) have been modified by adding the term “free L-(+)-glutamic
acid”;
— in Clause 4, the description of “extraction of L-(+)-glutamic acid of test portion” has been modified
and the detection wavelength has been changed from “492 nm” to “490 nm”;
— in 7.1, the identification of enzyme activity units for diaphorase and glutamate dehydrogenase has
been supplemented; the concentration of KOH, NAD has been modified; the NAD and diaphorase
have been mixed into a solution; and the buffer, NAD and enzymes have been labelled with R1, R2,
and R3;
— the apparatus list (7.2) has been updated;
— in 7.3, the procedure of the test method of spectrophotometer has been modified by halving the
sample mass and solution volume;
— in 7.3.4, the method of judging the absorbance of the reaction end point has been modified and, as a
result, the previous Annex B “Example of plotting and extrapolation of absorbance values” has been
deleted;
iv © ISO 2021 – All rights reserved

— in 8.4, the formula and symbol description of spectrophotometer has been modified;
— the previous Annex C “Derivation of equation for calculation of L-(+)-glutamic acid content” has
been deleted;
— the Bibliography has been updated.
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.
INTERNATIONAL STANDARD ISO 4134:2021(E)
Meat and meat products — Determination of L-(+)-
glutamic acid content — Reference method
1 Scope
This document specifies the spectrophotometer method and the light absorption microplate reader
method for the determination of the free L-(+)-glutamic acid content of meat and meat products.
This document is applicable to meat and meat products, including livestock and poultry products.
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 648, Laboratory glassware — Single-volume pipettes
ISO 1042, Laboratory glassware — One-mark volumetric flasks
ISO 1442, Meat and meat products — Determination of moisture content (Reference method)
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 8655-2, Piston-operated volumetric apparatus — Part 2: piston pipettes
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
free L-(+)-glutamic acid
L-(+)-glutamic acid and glutamate existing in meat and meat products in the form of free state
4 Principle
The free L-(+)-glutamic acid present in a test portion is extracted with perchloric acid solution. The
extract is centrifuged, decanted and filtered, and diluted to appropriate concentration with water, and
the pH is adjusted to 10. Nicotinamide adenine dinucleotide (NAD) is reduced by the L-(+)-glutamic
acid in the presence of glutamate dehydrogenase, see Formula (1). The resultant reduced nicotinamide
adenine dinucleotide (NADH) reacts with iodonitrotetrazolium chloride in the presence of diaphorase,
see Formula (2). The resulting formazane is measured at a wavelength of 490 nm and the free L-(+)-
glutamic acid content of the test sample is calculated.
glutamatedehydrogenasee
+
Lg--()++lutamicacid/glutamateNAD +HO←→
(1)
+
α-ketoglutamate++NADH NH +H
diaphorase
+ +
NADH + iodonitrotetrazolium chloride +H ←→ NAD + formazane (2)
5 Sampling
Sampling is not part of the method specified in this document. A recommended sampling method is
given in CAC/GL 50-2004.
It is important that the sample received by the laboratory 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 at a temperature of 4 °C or frozen
at −18 °C if not immediately analysed, so that deterioration and change in composition are prevented.
6 Preparation of test sample
Homogenize the laboratory sample with the appropriate equipment (see 7.2.1). Note that the
temperature of the sample should not exceed 25 °C. If a mincer is used, process the sample at least twice
with the equipment.
Fill a suitable airtight container with the prepared sample. Seal the container and store it at a
temperature of 4 °C or frozen at −18 °C if not immediately analysed, so that deterioration and change
in composition are prevented. Analyse the sample as soon as practicable, but always within 24 h after
homogenization.
7 Test method of spectrophotometer
7.1 Reagents
Only reagents of recognized analytical grade and only water of at least grade 2 purity as defined in
ISO 3696 shall be used. Except for the solutions of inorganic compounds (7.1.1 and 7.1.2), store all
solutions in stoppered brown glass bottles which have been scrupulously cleaned and steamed or
sterilized.
7.1.1 Dilute perchloric acid, c = 1,0 mol/l.
WARNING — Contact with oxidizable or combustible materials or with dehydrating or reducing
agents can result in fire or explosion. Persons using this acid should be thoroughly familiar with
its hazards. See the safety practices listed in Annex A.
Add 8,6 ml of the perchloric acid (70 g/ 100 g, ρ = 1,67 g/ml) to the bulk of water, diluting to 100 ml.
7.1.2 Potassium hydroxide solution, c = 4 mol/l, 2 mol/l, 0,5 mol/l and 0,02 mol/l.
Dissolve 22,4 g of potassium hydroxide in water. Dilute the solution to 100 ml, c = 4 mol/l, and mix
evenly after cooling.
Dissolve 11,2 g of potassium hydroxide in water. Dilute the solution to 100 ml, c = 2 mol/l, and mix
evenly after cooling.
Transfer 2,5 ml of 2 mol/l potassium hydroxide solution to 10 ml volumetric flask, dilute to the mark
with water and mix, c = 0,5 mol/l.
Transfer 0,1 ml of 2 mol/l potassium hydroxide solution to 10 ml volumetric flask, dilute to the mark
with water and mix, c = 0,02 mol/l.
2 © ISO 2021 – All rights reserved

7.1.3 Solution R1, triethanolamine phosphate buffer solution, pH = 8,6.
Dissolve 1,86 g of triethanolamine hydrochloride in approximately 25 ml of water, adjust the pH to 8,6
with 2 mol/l potassium hydroxide solution (7.1.2), detecting with a pH-meter. Add 0,68 g of octylphenol
decaethyleneglycol ether (e.g. Triton X-100). Dilute to 100 ml with water and mix (solution A).
Dissolve 0,86 g of dipotassium hydrogen phosphate (K HPO ) and 7 mg of potassium dihydrogen
2 4
phosphate (KH PO ) in water. Dilute to 100 ml with water and mix evenly (solution B).
2 4
Mix 20 ml of solution A with 5 ml of solution B.
The solution is stable for two months when stored at a temperature of between 0 °C and 6 °C.
1)
7.1.4 Solution R2, the mixed solution of NAD and diaphorase (lipoamide dehydrogenase EC
1.8.1.4), ρ = 11 mg/ml, diaphorase, approximately 4 IU/ml.
NAD
Weigh 110 mg of NAD, and approximately 8 mg (approximately 40 IU) of diaphorase into a stoppered
flask. Add 10,0 ml water and mix evenly.
The solution is stable for one week when stored in the dark at a temperature of between 0 °C and 6 °C.
7.1.5 Solution R3, iodonitrotetrazolium chloride (INT) solution, 2-(4-iodophenyl)-3-(4-
nitrophenyl)-5-phenyltetrazolium chloride, ρ = 0,6 mg/ml.
Weigh 6 mg of INT into a small, stoppered brown flask. Add 10 ml of water and mix evenly.
The solution is stable for four weeks when stored in the dark at a temperature of between 0 °C and 6 °C.
7.1.6 Solution R123, the mixed solution of solution R1, solution R2 and solution R3.
Pipette 6 ml of solution R1, 2 ml of solution R2 and 2 ml of solution R3 into a stoppered brown glass
bottle, and mix evenly before the test.
The mixed solution is stable for 1 h in stoppered brown glass bottles at room temperature.
7.1.7 Glutamate dehydrogenase (GLDH) solution (EC 1.4.1.3), approximately 900 IU/ml.
Weigh 10 mg (approximately 900 IU) of lyophilized glutamate dehydrogenase (GLDH) into a small
stoppered flask. Add 1 ml water and mix.
Insulated from ammonium sulfate, ethylene-dinitrilotetraacetic acid (EDTA) and glutaminase, this
solution is stable for 12 months when stored at a temperature between 0 °C and 6 °C.
7.1.8 L-(+)-glutamic acid standard stock solution, ρ = 1 000 mg/l.
Weigh, to the nearest 0,000 1 g, approximately 50,0 mg of L-(+)-glutamic acid (C H O N). Dissolve it in
5 9 4
approximately 25 ml of water.
Adjust the pH to a value range from 5 to 6 with a few drops of 2 mol/l potassium hydroxide solution
(7.1.2). Then adjust the pH to 7,0 slowly with 0,02 mol/l potassium hydroxide solution (7.1.2). Dilute to
50 ml with water and mix evenly.
The solution is stable for six months when stored at a temperature between 0 °C and 6 °C.
1) The EC number refers to the Enzyme Classification number as given in: The International Union of Biochemistry,
Enzymenomenclature, Elsevier, Amsterdam, 1965.
7.1.9 L-(+)-glutamic acid standard solution, ρ = 100 mg/l.
Pipette accurately 5,0 ml of L-(+)-glutamic acid standard stock solution (7.1.8) into a 50 ml volumetric
flask (7.2.8), dilute to the mark with water and mix evenly.
The solution is for the current use.
7.1.10 L-(+)-glutamic acid series standard solution, ρ = 5 mg/l, 10 mg/l, 15 mg/l, 20 mg/l, 30 mg/l
and 40 mg/l.
Pipette accurately 0,50 ml, 1,00 ml, 1,50 ml, 2,00 ml, 3,00 ml and 4,00 ml of L-(+)-glutamic acid standard
solution (7.1.9) into each of six 10 ml volumetric flasks (7.2.8) separately, dilute to the mark with water
and mix evenly.
The solution is for the current use.
7.2 Apparatus
The usual laboratory equipment and, in particular, the following shall be used.
7.2.1 Mechanical or electrical equipment, capable of homogenizing the lab
...